Axonal growth inhibitors and their receptors in spinal cord injury: from biology to clinical translation

Abstract

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelin-associated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19 (that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the RhoA/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Introduction

In the adult mammalian nervous system, following a traumatic spinal cord injury (SCI), neurons are unable to regenerate synaptic contacts due to the reduced inherent ability of the central nervous system (CNS), the presence of extrinsic factors that inhibit growth and reduced levels of correct growth factors (Zheng and Tuszynski, 2023). Immediately after trauma, damaged nervous tissue undergoes a series of immune, inflammatory and apoptotic events. The inflammatory response is mediated by invading macrophages, resident microglia, and oligodendrocyte progenitor cells (OPCs) that, together with damaged neurons, astrocytes, and myelin sheaths, release myelin-associated inhibitors and chondroitin sulfate proteoglycans (CSPGs) (Filbin, 2003; Boghdadi et al., 2018). The “scar” formed at the injury site seals the damaged tissue, creating an astrocytic outer border that minimizes secondary injury (Sofroniew, 2015). Myelin-associated inhibitors and CSPGs inhibit axonal regeneration upon binding to an array of molecular receptors and, together with their downstream effectors, compromise post-SCI tissue repair. Because regeneration of damaged circuits is blocked, SCI results in marked alterations in sensory, motor, and autonomic function. The quality of life of patients is thus severely affected. The interruption of ascending and descending neuronal tracts and the development of alternative neuronal circuits independent from supraspinal input that result from spontaneous sprouting of spared axons, give rise to compensatory but oftentimes erroneous plasticity that results in events such as pain or bladder dysfunction. This review addresses current knowledge on interactions of inhibitory molecules of axonal growth with their receptors and the downstream signaling pathways that lead to failure to regenerate and the state of the art on the application of this knowledge to the clinics.

Search Strategy

Literature search was performed on PubMed database on inhibitory molecules of axonal growth after SCI, their receptors and state of the art on the application of this knowledge to animal models and the clinics to treat spinal cord injuries. Original search was conducted between September and December 2022 to include progress in this field that spans at least the last 25 years.

Molecular Inhibitors of Axonal Growth

Myelin-associated inhibitors of axonal growth

Inhibition of axon outgrowth by CNS myelin has been demonstrated decades ago (Schwab and Thoenen, 1985; Caroni and Schwab, 1988a; Savio and Schwab, 1989) and early studies showed the presence of specific proteins in the myelin sheath with inhibitory properties for sprouting axons. While some remain to be identified, neurite outgrowth inhibitor A (Nogo-A), myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp) are now widely known as strong blockers of neurite outgrowth.

Nogo-A

Nogo is a member of the reticulon (Rtn)-family of membrane proteins, expressed in three isoforms: Nogo-A, -B, and -C. These isoforms are derived from the alternate promoter and splice usage but exhibit a common C-terminal domain related to the Rtn-family of proteins (GrandPré et al., 2000; Oertle and Schwab, 2003; Oertle et al., 2003). Nogo-A (Rtn4A) is expressed in oligodendrocytes that surround myelinated axons in the adult CNS (Caroni and Schwab, 1988a, b) and subpopulations of central and peripheral neurons (Chen et al., 2000). Nogo-A is expressed by the endoplasmic reticulum and cell membrane (Schwab, 2010) and encompasses two extracellular inhibitory domains: Nogo-66 and Nogo-Δ20. Nogo-66 interacts with Nogo receptor 1 (NgR1) (Fournier et al., 2001), but due to the lack of an intracellular domain, NgR1 forms a receptor complex with leucine-rich repeat and immunoglobulin-like domain-containing NgR interacting protein 1 (Lingo-1) (Mi et al., 2004) and p75 (McMahon et al., 1994) and/or TROY (Tnfrs19) (Park et al., 2005; Shao et al., 2005). Nogo-66 can also bind to the paired immunoglobulin-like receptor B (Pir-B) (Atwal et al., 2008). Nogo-Δ20 binds to membrane protein tetraspanin-3 (Thiede-Stan et al., 2015), which co-clusters with sphingosine-1 phosphate receptor 2 (Kempf et al., 2014; Figure 1).

Figure 1.

Molecular mechanisms of inhibitory and repulsive molecules signaling.

Myelin-associated inhibitors neurite outgrowth inhibitor A (Nogo-A), through its Nogo-66 inhibitory domain, oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG) collectively signal through the receptor complexes Nogo receptor 1 (NgR1), leucine-rich repeat and Ig domain containing 1 (Lingo-1) and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19 (p75/TROY), and through the NgR1 and paired immunoglobulin-like receptor B (Pir-B). MAG can also signal through the NgR1 homolog Nogo receptor 2 (NgR2) and via the gangliosides GD1a and GT1b. Additionally, Nogo-66 signals through the NgR1, PlexinA2, and Collapsin response mediator protein 2 (CRMP2). Nogo-Δ20, another Nogo-A inhibitory domain, signals through the receptor complex composed of Tetraspanin 3 (TSPAN3), Sphingosine-1-Phosphate Receptor 2 (S1PR2), and the syndecans SDG3 and SDG4, either via Leukemia-associated Rho guanine nucleotide exchange factor (LARG) or focal adhesion kinase (FAK) signaling, both converging to the Ras Homolog Family Member A/Rho-associated protein kinase (RhoA/ROCK) signaling pathway. NgR1, together with Lingo-1 and p75/Troy signals through RhoA/ROCK, LIM kinase (LIMK), and Cofilin, the same signaling pathway followed by PlexinA2, NgR1, and CRMP2 complex. NgR1 and Pir-B signaling can activate plenty of sarcoma (src) homology domain 3 (SH3) (POSH) through an unknown mechanism. POSH, in its turn, can activate the RhoA/ROCK signaling through Shroom or can activate a kinase cascade via leucine zipper kinase (LZK), mitogen-activated protein (MAP) kinase kinase (MKK), Jun N-terminal kinase (JNK) and Myosin II. LOTUS, an endogenous NgR1 inhibitor, can bind to this receptor and suppress axonal growth inhibition. Chondroitin sulfate proteoglycans (CSPGs) can induce growth inhibition by binding to NgR1 and NgR3 receptors, as well as receptor protein tyrosine phosphatase σ (RPTPσ), and leucocyte common antigen-related (LAR). When activated, these receptors activate the RhoA/ROCK signaling pathway and, in the case of RPTPσ and LAR, inactivate Akt and Erk pathways to inhibit axonal growth. When the repulsive molecule RGMa binds to its receptor neogenin, the RhoA/ROCK pathway can be activated through LARG activation; in parallel, RGMa/neogenin signaling can go inhibit FAK and p120GAP and also induce actin depolymerization and inhibition of neuronal outgrowth. Created with Adobe Illustrator 2021.

In vivo studies using antibodies to block Nogo-A in rodents were initiated decades ago. Anti-Nogo-A antibody therapies induced the regeneration of damaged tracts (Schnell and Schwab, 1990; Brösamle et al., 2000) and improved sensory motor function after SCI (Schnell and Schwab, 1990, 1993; Bregman et al., 1995; Z’Graggen et al., 1998; Raineteau et al., 1999; Merkler et al., 2001; Liebscher et al., 2005; Freund et al., 2006, 2007). Anti-Nogo-A antibodies are also effective in restoring autonomous bladder voiding in SCI rats (Liebscher et al., 2005). More recently, work from Schwab’s lab showed that the application of anti-Nogo-A antibodies to rats with incomplete SCI prevented the development of urinary dysfunction, in particular, detrusor-sphincter dyssynergia (Schneider et al., 2019; Sartori et al., 2020). The same effects, however, were not visible in animals with complete spinal cord transections, highlighting the importance of spared descending fibers in the reestablishment of lower urinary tract circuits. The effects of anti-Nogo-A therapy were also demonstrated in non-human primates (Fouad et al., 2004; Freund et al., 2007, 2009; Hoogewoud et al., 2013). Moreover, work from Ichiyama’s lab showed a promising therapeutic approach for SCI treatment, the combination of anti-Nogo-A antibody administration with intense locomotor training. This leads to heightened recovery of locomotor functions, compared to single interventions (Chen et al., 2017). The clinical combined application of anti-Nogo-A antibodies and physical rehabilitation for SCI treatment is currently being investigated in a multicentric, European, randomized, double-blind, and placebo-controlled phase II trial (Sartori et al., 2020), and it will be discussed ahead.

MAG

MAG is a type-1 transmembrane protein and a member of the Siglec family of sialic acid binding proteins and the first myelin inhibitor to be identified (McKerracher et al., 1994; Mukhopadhyay et al., 1994). MAG comprises two isoforms: large (L)-MAG, mainly expressed in the peripheral nervous system, and small (S)-MAG, predominant in the CNS (Salzer et al., 1987; Miescher et al., 1997). MAG is expressed by myelinating glia, peripheral nervous system Schwann cells, and CNS oligodendrocytes (Quarles, 2007). It plays a dual role according to the developmental stage, as MAG regulates developmental axonal growth (Johnson et al., 1989; Turnley and Bartlett, 1998) but neurite outgrowth of adult neurons is inhibited by MAG (McKerracher et al., 1994; Mukhopadhyay et al., 1994; Shen et al., 1998). This switch is age- and neuron type-dependent (DeBellard et al., 1996; Shen et al., 1998) and makes MAG the only myelin inhibitor that promotes growth during development. MAG exerts its effect by binding independently to several receptors, including the GPI-anchored NgR1 and its homolog Nogo receptor 2 (NgR2) (Venkatesh et al., 2005). However, NgR receptors appear to play a minor role in outgrowth inhibition by MAG, as sensory neurons from NgR1/2 double knockout mice exposed to MAG are still moderately inhibited (Wörter et al., 2009), indicating that MAG uses alternative receptors. These include neuronal gangliosides GD1a and GT1b (Yang et al., 1996; Vyas et al., 2002; Vinson et al., 2003), to which MAG binds independently, as well as Pir-B (Atwal et al., 2008), β1-integrin (Goh et al., 2008) and low-density lipoprotein receptor-related protein 1 (Stiles et al., 2013; Figure 1). After SCI, Mag-null mice show no enhancement of axonal growth (Bartsch, 1996), an observation later confirmed by Strittmatter’s lab. They showed that Mag deletion does not affect axonal growth or locomotion (Cafferty et al., 2010). Zheng’s lab also showed that even though Mag knockout mice exhibit sprouting of serotonergic fibers at the lumbar enlargement below the level of hemisection injury, deleting MAG in both hemisection and pyramidotomy models decreases corticospinal tract (CST) sprouting and does not promote its regeneration (Lee et al., 2010b). These findings suggest that MAG may play some sort of neuroprotective role after CNS injury (Lee et al., 2010b; Silver, 2010). However, the lack of published studies on the role of MAG in CNS regeneration precludes the true understanding of MAG function.

OMgp

OMgp is a member of the leucine-rich repeat protein family. It is a membrane-bound protein, connecting to the cell surface via a GPI-anchor. OMgp is expressed by oligodendrocytes, mature CNS neurons, in the glial-axonal border of myelinated axons in the brain and spinal cord (Vourc’h and Andres, 2004; Lee et al., 2009) and by astrocytes (Zhang et al., 2014). Like Nogo-A and MAG, OMgp binds to NgR1/Lingo-1/p75 and/or TROY receptor complex and Pir-B (Wang et al., 2002b; Atwal et al., 2008; Figure 1).

Much less is known about OMgp, but studies have demonstrated its strong inhibitory properties on axonal growth (Kottis et al., 2002; Wang et al., 2002b). Early evidence suggested in OMgp-null mice there is collateral sprouting at Ranvier nodes (Huang et al., 2005), highlighting the role of OMgp in the integrity of the myelin-axon interface. However, more recent studies reported contrary findings, evidencing the presence of OMgp at the surface of mature myelinated axons but excluded it from compact myelin and nodes (Chang et al., 2010). After SCI, OMgp is upregulated at the injury site (Guo et al., 2007) and involved in neurite outgrowth inhibition when it synergizes with MAG and Nogo-A (Ji et al., 2008; Cafferty et al., 2010). No alterations in OMgp expression are detected above and below the injury site (Guo et al., 2007; Chambel et al., 2022).

Scar-associated players of axonal growth

Following SCI, immune cells, microglia, and OPCs invade the injury site and recruited astrocytes become reactive and hypertrophic. The scar that subsequently forms has two main components: the lesion core with a fluid-filled cavity surrounded by fibroblasts, endothelial cells, and macrophages that give rise to a highly inhibitory environment for regeneration, the fibrotic scar; and an astrocytic border that seals the injury site, restricting migration of immune cells into the CNS and sparing non-damaged tissue, therefore allowing re-establishment of homeostasis (O’Shea et al., 2017; Zheng and Tuszynski, 2023). The true role of astrocytic borders is still not fully understood. In the past, astrocyte borders had been associated with axonal failure to regenerate after a CNS lesion, as reactive astrocytes secrete CSPGs, traditionally seen as important blockers of axonal regeneration (Silver and Miller, 2004). However, in the last few years, scar-forming astrocytes were shown to support post-SCI regeneration. When astrocytic scar formation is hindered, by preventing its formation, inhibiting scar-forming astrocytes or completely ablating chronic scars, transected axons in a mouse model of SCI are unable to regrow (Anderson et al., 2016), while the reverse occurs when growth factors are delivered (Anderson et al., 2016, 2018). The reason for this resides in the expression, by scar-forming astrocytes, of axon-growth supporting molecules, such as CSPG4 and CSPG5, previously thought to only have inhibitory properties (Anderson et al., 2016). Likewise, keratan sulfate proteoglycans, produced by macrophages and OPCs at the lesion site, and known to be highly inhibitory to regenerating axons (Jones et al., 2002), have recently been shown to have pro-regenerative properties. After complete spinal transection, the spiny mouse (Acomys cahirinus) exhibits both regeneration of several tracts and synapse formation due to the overexpression pro-regenerative keratan sulfate proteoglycans at the injury site, as well as lack of scar tissue at the lesion site (Nogueira-Rodrigues et al., 2022). Certain populations of neurons have also been demonstrated to express pro-regenerative genes in response to injury. Specifically, rodent spinocerebellar neurons express regeneration-associated genes (RAG), such as Atf3, Sox11, Sprr1a, and Gap43 in the lumbar cord after thoracic SCI (Matson et al., 2022). These lumbar cord RAG⁺ expressing neurons survive contusion, even increasing their numbers and evidencing branching and fiber reorganization in the thoracic cord (Matson et al., 2022). These findings break the widely accepted dogma that after a CNS injury, the CNS environment is exclusively inhibitory towards regrowth.

Guidance molecules, such as semaphorins, ephrins, and WNTs, also participate in post-SCI plasticity and become highly inhibitory, blocking regeneration. They are also important players during development, aiding growth cone navigation in embryonic environments by attracting or repelling growing axons. However, due to their complex roles and many potential interactions with both myelin and the extracellular matrix (Zheng and Tuszynski, 2023), their exact function is not fully understood. Here, we will only focus on CSPGs and RGMa. Further details on the role of guidance molecules can be found elsewhere (Giger et al., 2010; Hollis, 2016).

Chondroitin sulfate proteoglycans - CSPGs

Chondroitin sulfate proteoglycans are a diverse group of glycoproteins ubiquitously expressed in the extracellular matrix or cell surface (Maeda, 2015). CSPGs consist of core proteins to which one or more glycosaminoglycan chains are attached via serine residues. CSPGs are mainly synthesized by astrocytes and neurons, but also by OPCs and macrophages (Jones et al., 2002). CSPGs are named after their core proteins and are divided into lecticans (versican, neurocan, brevican, and aggrecan), phosphacan, neuron-glial antigen 2, and the small leucine-rich proteoglycans decorin and biglycan (Mironova and Giger, 2013; Bradbury and Burnside, 2019). In the normal CNS, CSPGs are expressed during development, regulating axon guidance, and in the adult, modulating synaptic connections and cell migration (Viapiano and Matthews, 2006). After CNS injury, CSPGs are upregulated and become abundant perilesionally and at distal spinal segments (Andrews et al., 2012; Chambel et al., 2022). They inhibit neurite outgrowth both in vitro (Jin et al., 2018) and in vivo conditions (Andrews et al., 2012), blocking axonal sprouting at the injury site and hindering the regeneration of damaged tracts. CSPGs inhibition of axonal growth requires activation of membrane-bound receptors including the receptor-type protein tyrosine phosphatase sigma (RPTPσ) (Shen et al., 2009), leukocyte common antigen-related phosphatase (LAR) (Fisher et al., 2011), NgR1 and Nogo receptor 3 (NgR3) (Dickendesher et al., 2012) (Figure 1).

The inhibitory nature of CSPGs in the CNS is highly variable, reflecting the type of injury, as well as the affected area. In traumatic lesions, CSPG expression can be observed at the injury sites as early as 24 hours and up to months post-injury. For example, in a rat model of cerebral cortex injury, OPCs and astrocyte-derived Versican and Neurocan are upregulated at 7 days-post injury (dpi) (Asher et al., 2000, 2002). After spinal contusion, at the lesion site, neurocan, brevican, and versican are elevated immediately after injury and peak at 14 dpi, with brevican remaining elevated for at least 2 months (Jones et al., 2003). Phosphacan expression in the lesion first decreases after injury to then slowly recover and peaking two months-post injury (Jones et al., 2003). The distribution of specific CSPGs around the lesion also varies. While neurocan and brevican are expressed near the lesion, phosphacan and versican are found further away (Jones et al., 2003). Upregulation of CSPGs at the lesion site has been well-documented and proven to hinder axonal regeneration, but alterations distant from the injury are also observed. An early work by Andrews et al. (2012) showed that after a T8 spinal contusion, aggrecan, neurocan, brevican, and neuron-glial antigen 2 were altered in distal lumbar and cervical enlargements. More recently, Chambel et al. (2022) demonstrated alterations in the expression of both phosphacan and neurocan at the lumbosacral cord in a rat model of thoracic spinal cord transection. Levels of both CSPGs increased 7 dpi and returned to baseline values 28 dpi. Despite the inhibitory properties of phosphacan and neurocan, sprouting of sensory bladder afferents still occurred within the lumbosacral cord, which has been linked to the emergence of bladder dysfunction.

Even though some evidence shows that CSPGs core proteins can inhibit neuronal growth (Ughrin et al., 2003), it is the accessory glycosaminoglycan chains the main responsibles for CSPG-induced blockade of axonal growth. Accordingly, degradation of glycosaminoglycan chains by chondroitinase ABC (ChABC), which exposes the protein core, promotes axonal sprouting and regain of function following SCI (Bradbury et al., 2002; Bartus et al., 2014; Muir et al., 2019; Rosenzweig et al., 2019; Day et al., 2020; Takiguchi et al., 2022). The beneficial effect of ChABC treatment has been firstly shown following in vivo administration (Bradbury et al., 2002), and further confirmed in neuronal cultures (Day et al., 2020) and several SCI models, such as contusion (Caggiano et al., 2005), compression (Novotna et al., 2011), crush (Barritt et al., 2006) and transection (Takiguchi et al., 2022) injuries. Its promising and safe use has also been tested in several species, such as mouse, rat, cat, a canine clinical model (Hu et al., 2018), and non-human primates (Rosenzweig et al., 2019). Approaches that combine ChABC with other experimental therapies are even more promising. In models of SCI, combining ChABC administration with neurotrophins (Tom et al., 2009; Lee et al., 2010a; García-Alías et al., 2011; Hunanyan et al., 2013; Kanno et al., 2014), growth factors (Karimi-Abdolrezaee et al., 2012; Lee et al., 2013; Tom et al., 2013; Zhang et al., 2013; Alluin et al., 2014; DePaul et al., 2017), rehabilitation (Wang et al., 2011a; Zhao et al., 2013; Alluin et al., 2014; Hu et al., 2018; Prager et al., 2021), stem cell therapy (Sarveazad et al., 2017; Suzuki et al., 2017; Buzoianu-Anguiano et al., 2020; Jevans et al., 2021), antibodies against Nogo-A (Zhang et al., 2013; Zhao et al., 2013), and various others (Bai et al., 2010; Mountney et al., 2013; Grosso et al., 2014; Janzadeh et al., 2017; Wu et al., 2017; Xia et al., 2017; Liu et al., 2018; Lu et al., 2020) shows enhanced axonal regeneration and functional recovery (locomotor or hand coordination), in comparison with single therapies.

In earlier works, ChABC was delivered intrathecally (Bradbury et al., 2002), but this enzyme was unstable at 37°C. Better results were obtained using other delivery strategies, including synthetic scaffolds or stabilized ChABC, with delivery using lentivirus and adeno-associated viral vectors currently being the most successful and used approach. These vectors can provide stable and long-term expression of ChABC in transduced cells at the injury site (Muir et al., 2019), as well as CSPG digestion over a large area of the cord (Bartus et al., 2014). Expression of ChABC transgenes by adeno-associated viral vectors begins 1 week after delivery and reaches maximal expression at 3 weeks; lentiviral vectors are able to start transgene expression 24-48 hours post-injury. These differences in expression of ChABC via viral vectors allow differential SCI treatment at different stages. In fact, timing and duration of ChABC administration seem to be detrimental for gain-of-function after SCI. Intrathecal administration of high doses of ChABC at the same time of SCI seems to produce nefarious effects (Cheng et al., 2015), including subarachnoid bleeding and animal death, not seen after sub-acute administrations. High-dosage intrathecal administration after spinal transection (Cheng et al., 2015; Janzadeh et al., 2017; Liu et al., 2018) or long-term viral vector delivery after spinal contusion (Bartus et al., 2014; James et al., 2015) result in axonal growth and functional recovery, making 48 hours–7 days following injury the ideal timeframe for ChABC intervention after SCI (Muir et al., 2019). For the optimal time of administration, it is necessary to consider an appropriate timeframe for tissue healing by scar formation and blood-brain barrier leakage (Rolls et al., 2009). Scar tissue, however, should not be fully consolidated. Studies in SCI dogs and monkeys (Hu et al., 2018; Rosenzweig et al., 2019), as well as in humans to treat disc herniation (Chiba et al., 2018) have proven that ChABC is effective and safe, without adverse effects and producing enhanced recovery in treated subjects, and thus evidencing the potential use of ChABC for SCI treatment.

In addition to preventing axonal expansion necessary to repair injured axons, recent evidence highlighted a pro-inflammatory post-SCI role for CSPGs, exacerbating the immune response (Bartus et al., 2014; Didangelos et al., 2014; Dyck et al., 2018). Bradbury’s lab showed the involvement of CSPGs in macroglia, macrophages, and T-lymphocytes modulation, preventing the conversion of immune cells at the injury site from a pro-inflammatory to a reparative phenotype. A pro-reparative phenotype can be achieved by lentiviral ChABC digestion, leading to attenuation of the pro-inflammatory environment and immune cell clearance (Francos-Quijorna et al., 2022). These findings highlight the dynamic role CSPGs play in both axon growth and inflammation following SCI and open the doors to immunomodulatory therapies that target both complications.

Repulsive guidance molecule A - RGMa

Repulsive guidance molecule A (RGMa) is part of the repulsive guidance molecule family. RGMa is present in myelin and accumulates in the glial scar after SCI (Schwab et al., 2005), where it is expressed in neurons, oligodendrocytes, astrocytes, activated microglia, and macrophages (Mothe et al., 2017). RGMa binds to its neuronal receptor, neogenin (Rajagopalan et al., 2004), inhibiting neurite outgrowth (Tassew et al., 2012; Yamashita, 2019; Figure 1). RGMa blockade promotes regeneration, plasticity, and recovery following SCI in rats (Hata et al., 2006; Mothe et al., 2017, 2020), mice (Nakanishi et al., 2019) and non-human primates (Nakagawa et al., 2019).

Receptors of Molecular Inhibitors of Axonal Outgrowth in Spinal Cord Injury

NgR1

Myelin inhibitors are known to induce growth cone collapse after SCI via activation of their surface receptors. This was demonstrated following the identification of NgR1 as a high-affinity receptor for both Nogo-A, MAG, and OMgp (Fournier et al., 2001; Domeniconi et al., 2002; Liu et al., 2002; Wang et al., 2002b), as dorsal root ganglion neurons from NgR1-null mice do not suffer growth cone collapse when acutely exposed in vitro to soluble Nogo-A, MAG, and OMgp (Kim et al., 2004; Chivatakarn et al., 2007). In vivo, NgR1 deletion in SCI animals results in improved functional recovery and neuronal regeneration (Kim et al., 2004). Strittmatter’s lab developed a strategy to block NgR1 by creating a soluble truncated NgR1 fusion protein - NgR1(310)ecto-Fc (Fournier et al., 2002). Administration of this decoy proved to be effective in treating rats acutely (Li et al., 2004; Wang et al., 2006) and chronically (Wang et al., 2011b) after SCI, increasing axonal growth and functional recovery. Pre-clinical studies in non-human primates further demonstrated the efficacy of NgR-Fc decoy protein and a clinical trial in chronic cervical SCI patients is currently undergoing (see below). However, targeting Ngr1 to treat SCI might not be sufficient, as this GPI-anchored receptor requires other co-receptors, such as p75 and TROY, to initiate signal transduction.

p75 and TROY

The function of p75 is complex and difficult to discern. When by itself or in association with Trk receptors, p75 can promote cell survival and nerve regeneration, but its activation has also been associated with cell death (Roux and Barker, 2002). Furthermore, p75 is known to co-localize with NgR1 and Pir-B, indicating its role in signal transduction of myelin inhibitors (Wang et al., 2002a; Wong et al., 2002; Fujita et al., 2011). In a mouse model of spinal contusion, p75 was shown to be essential for neuronal cell survival and locomotor recovery (Chu et al., 2007). However, genetic deletion or blockade with Fc-fusion protein promoted axonal regeneration after SCI in mice (Song et al., 2004), suggesting that p75 signaling could be compensated by other co-receptors. Another study used a different Fc-fusion protein, which also blocked p75. In the same model, this resulted in axonal regeneration, with decreased RhoA activation, and functional recovery (Wang et al., 2015). These conflicting results could be explained by different experimental time points for treatment delivery (2 versus 6 weeks post-injury) and might also reveal species-dependent differences. In contrast, the role of p75 signaling is rather clear in urinary dysfunction after SCI. Systemic administration to mice of LM11A-31, an agent that blocks activation of p75 by nerve growth factor precursor, reduced bladder pressure and hyperreflexia, increased bladder capacity, and led to earlier development of automatic micturition (Ryu et al., 2018). LM11A-31 also prevented urothelial hyperplasia and detrusor hypertrophy (Ryu et al., 2018). Similar results were obtained by Zabbarova et al. (2018). In this case, LM11A-31 ameliorated neurogenic detrusor overactivity and detrusor-sphincter dyssynergia, leading to greater voided volumes, which was also accompanied by preservation of the urothelial layer.

TROY is a functional homolog of p75 and was independently identified by two groups (Park et al., 2005; Shao et al., 2005). Neurons from Troy-null mice are less sensitive to myelin inhibitors (Shao et al., 2005) and TROY negatively modulates CNS remyelination. When OPCs submitted to Troy knockdown were transplanted into the spinal cord of SCI rats, increased remyelination and recovery were observed (Sun et al., 2014), confirming the role of TROY in blocking axonal growth.

Lingo-1

Lingo-1 is an element of the NgR1/p75 and/or TROY receptor complex (Mi et al., 2004). Lingo-1 activation appears to be critical for CNS myelination, inhibiting oligodendrocyte differentiation and myelination in mice (Jepson et al., 2012) and zebrafish (Yin and Hu, 2014). Lingo-1 blockade leads to increased myelination competence (Mi et al., 2005) while its blockade with a Lingo-1-Fc protein improved neuronal and oligodendrocyte survival, and axonal sprouting in SCI (Ji et al., 2006). Interestingly, after thoracic SCI in the rat, Lingo-1 levels are downregulated in lumbosacral sensory neurons that undergo intense sprouting into the superficial layers of the spinal cord, possibly in response to increased nerve growth factor levels (Chambel et al., 2022), which concurs with previous reports (Lee et al., 2007).

CRMP2 and PlexinA2

Recently, a novel NgR1 molecular partner has been identified. Collapsin response mediator protein 2 (CRMP2) is a CNS ubiquitously expressed microtubule-associated protein, expressed at pre- and post-synaptic sites. CRMP proteins mediate Semaphorin signaling through Plexin-containing receptors (Schmidt and Strittmatter, 2007). The Strittmatter lab has shown that CRMP2 physically associates with NgR1 and PlexinA2 into a signaling complex, a process mediated by Nogo-A (Sekine et al., 2019). This work demonstrated that the NgR1/PlexinA2/CRMP pathway is required to mediate Nogo-A induced inhibition of cortical neuron regeneration and CST sprouting. Accordingly, after SCI, CRMP2 knock-in (CRMP2KI) mice display improved SCI pathophysiology, exhibiting microtubule stabilization in neurons (Sugeno et al., 2021).

Pir-B

Pir-B is a receptor for Nogo-A, MAG, and OMgp (Atwal et al., 2008). Genetic ablation or pharmacological blockage of Pir-B in vitro promotes neurite outgrowth on substrates rich in myelin inhibitors or CNS myelin (Atwal et al., 2008). After SCI, Pir-B is overexpressed in dorsal root ganglion neurons, sciatic nerves, and spinal cord segments (Peng et al., 2015). Like NgR1, Pir-B can associate with p75 and mediate inhibition of axon growth (Fujita et al., 2011).

NgR3, LAR, and RPTPσ

NgR3, LAR, and RPTPσ are the exclusive high-affinity receptors for CSPGs. Several studies using genetic ablation of these receptors demonstrated their role in regulating axonal growth. Dickendesher et al. (2012) reported that CSPGs are able to bind to NgR1 and NgR3 with high affinity, resulting in inhibition of axonal growth. After genetic deletion of NgR3 and NgR1, there is no inhibitory effect of CSPGs and the optic nerve regenerates after crush injury in mice. Moreover, these authors also showed enhancement of regeneration of optic nerves genetic inactivation of RPTPσ in NgR1- and NgR3-null mice (Dickendesher et al., 2012), further demonstrating the role of RPTPσ in CSPG-mediated inhibition.

The importance of RPTPσ and LAR in CSPG-mediated axonal inhibition has also been demonstrated in SCI models. Mice in which RPTPσ was genetically eliminated exhibit increased regrowth of sensory and CST axons into the scar and caudal spinal cord, respectively (Shen et al., 2009; Fry et al., 2010). Lar-null mice showed improved locomotor function and sprouting of serotonergic and CST axons after SCI (Xu et al., 2015). Deleting genes encoding RPTPσ and LAR in adult neuronal cultures produces synergistic enhancements of axon growth (Ohtake et al., 2016). Furthermore, pharmacological inhibition of RPTPσ and LAR signaling has been shown to improve functional outcomes and sprouting of serotonergic fibers in spinal cord injured rats (Xie et al., 2006; Fisher et al., 2011; Lang et al., 2015) and to promote recovery of diaphragm function after cervical SCI (Urban et al., 2020; Cheng et al., 2021).

Downstream Signaling Pathways in Spinal Cord Injury

Following SCI, when growth cones contact with inhibitory molecules, their cognate receptors are activated. These interactions initiate downstream signaling pathways involved in cytoskeletal dynamics, the most well studied of which is the RhoA/Rho kinase (ROCK) pathway. When activated, RhoA/ROCK promotes F-actin disassembly in growth cones and hinders axon elongation, by preventing microtubule recruitment, which ultimately results in growth cone collapse (Maekawa et al., 1999; Wu et al., 2005). When active, Rho-GTP activates its downstream effector, ROCK. In turn, activated ROCK phosphorylates several downstream effectors, such as LIM kinase, CRMP2, adducin, and myosin light chain, among others, eventually leading to growth cone collapse and neurite outgrowth inhibition (Boghdadi et al., 2018; Kimura et al., 2021). NgR1-mediated signaling pathway includes RhoA, ROCK, LIM kinase, and Cofilin, ultimately resulting in actin depolymerization (Hsieh et al., 2006). Pir-B signals through different but interconnected downstream effectors that are able to crosstalk. These include plenty of sarcoma homology domain 3, that either converges with the ROCK pathway; or activates the leucine zipper kinase, mitogen-activated protein kinase kinase, Jun N-terminal kinase and Myosin II, eventually resulting in inhibition of axonal growth (Taylor et al., 2008; Gou et al., 2014; Boghdadi et al., 2018; Figure 1). CSPG activation of RPTPσ and LAR results in downstream activation of RhoA/ROCK and blockade of Akt and ERK cascades (Ohtake et al., 2016), as described in Figure 1.

Myelin inhibitors and CSPGs share several common downstream effectors that can potentially crosstalk and compensate for one another, hence presenting as potential therapeutic targets for SCI treatment. RhoA inhibition has attracted considerable attention and C3 transferase, obtained from Clostridium botulinum, has been shown to selectively inhibit RhoA. Tested in several pre-clinical studies, RhoA inactivation results in axonal sprouting and recovery of locomotor function after SCI (Dergham et al., 2002; Dubreuil et al., 2003), by reversing Rho activation and preventing p75-dependent cell death. The clinical application of C3 transferase, cethrin, is discussed ahead.

Therapeutic Modulation of Inhibitory Molecules for Spinal Cord Injury Treatment

Administration of anti-Nogo-A antibody in animal models of SCI has been shown to induce neural repair in rodents (Liebscher et al., 2005; Craveiro et al., 2013) and non-human primates (Fouad et al., 2004; Freund et al., 2007, 2009; Hoogewoud et al., 2013). These results supported a phase I, open-label, multicenter study in human patients with severe, acute SCI, initiated in 2006 and concluded in 2011 (ClinicalTrials.gov; Identifier: NCT00406016). In this trial, intrathecal administration of Nogo-A antibody in the sub-acute phase improved functional deficits of patients (Kucher et al., 2018). In 2019, the NISCI (Nogo inhibition in SCI study, a multicenter, European, randomized, double-blind, and placebo-controlled phase II trial was launched to investigate the efficacy of early treatment initiation (within 28 days post-injury) via repeated injections of anti-Nogo-A antibodies in participants with acute, cervical spinal injuries (ClinicalTrials.gov; Identifier: NCT03935321). In addition to anti-Nogo-A therapy, patients received rehabilitation training for up to six months after injury. The NISCI study was closed in 2022, with more than 110 patients enrolled (nisci-2020.eu) and results are currently under assessment.

In 2019, the RESET (ReNetX safety efficacy and tolerability of AXER-204 for chronic SCI) study, a two-part clinical trial to assess safety, tolerability, pharmacokinetics, and efficacy of AXER-204, a human NgR-derived fusion protein was initiated. This compound acts as a soluble decoy/trap for Nogo-A, MAG and OMgp and is delivered by intrathecal lumbar infusion to patients with chronic (≥ 1 year) cervical spinal cord injuries (Clinical Trials.gov; Identifier: NCT03989440). In pre-clinical studies, Strittmatter’s group has demonstrated that AXER-204 promotes functional recovery and enhances axonal growth in rats with spinal contusion (Wang et al., 2014) and recovery of forelimb and corticospinal regeneration in non-human primates with hemisection injuries (Wang et al., 2020). The RESET trial is currently recruiting patients.

The ELASCI, a phase II randomized, double-blind, placebo-controlled proof of concept study was initiated in 2020 and aims to assess the safety and efficacy of systemic intravenous administration of elezanumab, a human anti-RGMa monoclonal antibody, in acute traumatic cervical spinal cord injuries (ClinicalTrials.gov; Identifier: NCT0495538). Pre-clinical studies using anti-RGMa antibody in rats with contusion/compression spinal injuries showed neuronal sparing and neuroplasticity, as well as improvements in bladder function (Mothe et al., 2017, 2020). In non-human primates, elezanumab administration in an acute stage promoted neuroprotection, neuroplasticity, and recovery of neuromotor function after a hemicompression thoracic spinal injury (Jacobson et al., 2021). The ELASCI trial is currently in the recruitment stage.

Another promising drug to treat subjects with acute traumatic cervical spinal cord injuries that reached a phase 2b/3, double-blind, randomized, placebo controlled, multicenter study, was VX-210, (previously known as BA-210 and cethrin), a Rho-GTPase antagonist (ClinicalTrials.gov; Identifier: NCT02669849) (Fehlings et al., 2018). VX-210 proved to improve locomotor function and decrease lesion extension in rodents after spinal contusion (Lord-Fontaine et al., 2008). Even though VX-210 was well-tolerated by human subjects, the study ended prematurely when it failed to meet the primary efficacy endpoint. The study’s final analysis revealed no statistically significant differences between the VX-210 and placebo groups (Fehlings et al., 2021).

Conclusion

The adult mammalian CNS is unable to regenerate after injury. Consensus on axonal regeneration following SCI seems to lay on three major requirements: activation of neural intrinsic growth, to allow neurons to regenerate their axons following injury; modulation of neuron-extrinsic factors, such as myelin- and scar-derived inhibitors, creating both a permissive and supportive environment for axonal regrowth; and availability of specific chemoatractive growth factors, for neuronal survival and axon regeneration (Courtine and Sofroniew, 2019; Zheng and Tuszynski, 2023).

Pre-clinical and clinical investigation focused, for many years, on extrinsic factors, with the development of numerous pharmacological approaches to “inhibit the inhibitors” and promote repair. Targeting of myelin inhibitors in animal models and, more recently, in clinical trials, generates good results, with gain-of-function outcomes reported. However, these approaches mostly just induce plasticity and axonal sprouting rather than proper repair of the injured tracts and returning to pre-lesion conditions (Geoffroy and Zheng, 2014). While sprouting, defined as extension of new processes along the shaft on a spared axon, of uninjured distal axons occurs spontaneously after injury and allows new connections to be formed, neuronal circuits are usually not restored by these strategies and only replaced by new alternative pathways. This maladaptive sprouting leads to the emergence of unwanted, bothersome and dangerous conditions that emerge after SCI, as is the case of chronic central neuropathic pain, with nociceptive processes being activated in the absence of stimulation and/or amplified in the presence of a noxious stimulus (Deumens et al., 2008; Kang et al., 2020). Abnormal sprouting of peptidergic sensory afferents into the spinal cord, dependent on nerve growth factor, is also critically involved in development of autonomic dysreflexia (Cameron et al., 2006; Hou et al., 2008). Finally, nerve growth factor and brain-derived neurotrophic factor also regulate sprouting of bladder afferents, leading to urinary impairment (Yoshimura et al., 2006; Frias et al., 2015; Wada et al., 2018).

One of the issues of current clinical trials to treat SCI is the variable degree of success reported. These differences could be mitigated by back-translational approaches to fine-tune the animal models used in pre-clinical studies and their translational value in the clinics. For example, the use of rodents versus more clinically relevant models, such as non-human primates, should be explored. Many obstacles are still in the way of wider use of primates in research, mainly due to ethical, logistic, and cost-associated reasons. Advantages brought on by their use in pre-clinical studies could greatly improve the cost and outcomes in the clinic which would, ultimately, contribute to the advancement of SCI therapies. The type of injury used in pre-clinical studies should also be considered. Transection injuries are reproducible and usually accompanied by modest recovery; however, contusion models are more clinically relevant, but with severity of injury and functional outcomes being highly variable. Over the last few years, research has also shown that pre-clinical studies should aim to apply combinatorial therapies in the treatment of SCI. Extensive pre-clinical and clinical pharmacological approaches that target inhibitory molecules are delivering promising results and have the potential to produce breakthroughs in the treatment of CNS injuries. These treatments, however, would probably benefit from joint application with other supportive therapies, such as growth promoting or neurotrophic factors, stem-cell-based therapies and physical rehabilitation. Furthermore, neuroengineering therapies have proven, with great success, to produce extraordinary recovery outcomes after SCI (Capogrosso et al., 2016; Asboth et al., 2018; Bonizzato et al., 2018). Their synergistic use with already tested and hereafter established biological therapies should be the next great approach in the treatment of spinal cord injuries.

Additional file: Open peer review reports 1 ^{(96.2KB, pdf)} and 2 ^{(96.2KB, pdf)} .