New insights on the role of chondroitin sulfate proteoglycans in neural stem cell–mediated repair in spinal cord injury

Extensive neurodegeneration is a hallmark of traumatic spinal cord injury (SCI) that underlies permanent sensorimotor and autonomic impairments (Alizadeh et al., 2019). Following the primary impact, the spinal cord undergoes a cascade of secondary injury mechanisms that are driven by disruption of the blood–spinal cord barrier, vascular injury, glial reactivity, neuroinflammation, oxidative stress, lipid peroxidation, and glutamate excitotoxicity that culminate in neuronal and oligodendroglial cell death, demyelination, and axonal damage (Alizadeh et al., 2019). To achieve a meaningful functional recovery after SCI, regeneration of new neurons and oligodendrocytes and their successful growth and integration within the neural network are critical steps for reconstructing the damaged spinal cord tissue (Fischer et al., 2020). Neural precursor cells (NPCs) hold promises for repairing the damaged spinal network. NPCs are tissue-specific progenitors of the central nervous system with the multilineage potential to generate new neurons and glia (Fischer et al., 2020; Hosseini et al., 2024). However, extensive evidence indicates that an insufficient number of resident NPCs in the injured spinal cord and their predominant astrogenic fate hinder their capacity to replace damaged neurons following injury (Hosseini et al., 2024). Thus, transplantation of exogenous NPCs has been pursued as a more efficient approach to re-build the neuron–glia network within the injured spinal circuitry and improve functional recovery after SCI (Hosseini et al., 2024). NPC transplantation studies in the past two decades have revealed opportunities and challenges associated with this therapeutic approach. These studies have established that combinatorial strategies aimed at improving the injury microenvironment are beneficial to optimizing the long-term survival, migration, differentiation, and integration of engrafted NPCs after SCI (Karimi-Abdolrezaee et al., 2010; Hosseini et al., 2024). Scar formation and inhibitory modifications of the extracellular matrix in the post-SCI milieu are among the major mechanisms that hinder self-repair in the adult spinal cord (Alizadeh et al., 2019). Astrocytes and microglia respond to the injury robustly and in collaboration with pericytes and infiltrating meningeal cells form a fibrotic scar within and around the lesion (Alizadeh et al. 2019). Scar formation is a natural defense mechanism to limit the extent of the inflamed lesions at the early stage of SCI. However, over time the scar becomes a barrier to neural repair and regeneration. Upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) in the fibrotic scar is a major hallmark of SCI (Alizadeh et al., 2019). It is well-established that pathologic remodeling of CSPGs in the extracellular matrix and their long-lasting deposition in the lesion poses an inhibitory barrier to cell replacement (Karimi-Abdolrezaee et al., 2010; Hosseini et al. 2022) and axon regeneration (Lang et al., 2015) after SCI. We have shown that CSPGs inhibit survival, biodistribution, and neuronal differentiation of engrafted NPCs and drive their astrogenesis in SCI (Karimi-Abdolrezaee et al., 2010; Hosseini et al., 2022). Our study showed that degrading CSPGs using chondroitinase ABC was sufficient to enhance the regenerative response of engrafted NPCs and improve the functional recovery of rats with chronic SCI (Karimi-Abdolrezaee et al., 2010). This provided the proof-of-concept that targeting CSPGs is a promising strategy to optimize the outcome of NPC therapy for SCI (Karimi-Abdolrezaee et al., 2010; Hosseini et al., 2024). CSPGs mediate their effects primarily through two protein tyrosine phosphatase receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTP-σ) (Hosseini et al., 2024). We have shown that mouse and human NPCs highly express LAR and PTP-σ, and as such they can respond to the upregulated levels of CSPGs in their injury microenvironment (Dyck et al., 2015; Hosseini et al., 2022). In vitro studies revealed that exposure of NPCs to CSPGs restricts their regenerative properties including cell spreading, growth, survival, and proliferation through LAR and PTP-σ (Dyck et al., 2015; Hosseini et al., 2022). Importantly, activation of CSPG signaling drives the differentiation of NPCs towards an astrogenic fate and decreases the neurogenic and oligogenic capacity of NPCs (Dyck et al., 2019; Hosseini et al., 2022). Interestingly, we and others have also identified a role for CSPGs in promoting a pro-inflammatory response following injury (Dyck et al., 2018; Francos-Quijorna et al., 2022). We have shown that CSPGs elevate the pro-inflammatory phenotype of activated microglia, and suppressing LAR and PTP-σ promotes a wound-healing phenotype in these microglia that is associated with repair (Dyck et al., 2018). Pro-inflammatory microglia are known to inhibit neurogenesis and oligodendrogenesis in central nervous system injury and disease (Alizadeh et al. 2019). Thus, these findings unravel an indirect mechanism by which CSPGs block NPC differentiation in SCI. Mechanistically, CSPG/LAR/PTP-σ signaling regulates NPC functions by activating the Rho/ROCK (Rho-associated coiled coil containing protein kinase) pathway and inhibiting phosphorylation of Akt and Erk1/2 (extracellular signal-regulated kinase1/2) (Dyck et al., 2015; Figure 1). While blockade of each receptor individually reduces some of the inhibitory effects of CSPGs, their co-inhibition is needed to synergistically promote NPC properties under CSPG exposure (Dyck et al., 2015).Figure 1: Schematic figure describing mechanisms of CSPGs in regulating regenerative capacities of NPCs.SCI results in upregulation of CSPGs around the lesion site. Activation of the CSPG/LAR/PTP-σ axis reduces neurogenesis and oligogenesis of NPCs and restricts the regenerative capacity of NPCs including cell growth, survival, and proliferation by activating the Rho/ROCK pathway and decreasing phosphorylation of Akt and Erk1/2. Moreover, activation of CSPG/LAR/PTP-σ signaling decreases the neurogenesis of NPCs and increases their astrogenic fate by dysregulation of the Wnt/β-catenin pathway. Created with BioRender.com. CSPGs: Chondroitin sulfate proteoglycans; Erk1/2: extracellular signal–regulated kinase 1/2; LAR: leukocyte common antigen-related; NPCs: neural precursor cells; PTP: protein tyrosine phosphatase; SCI: spinal cord injury.Therapeutically, targeting LAR and PTP-σ receptors provides a feasible approach to overcoming CSPG effects. Although chondroitinase ABC is effective in removing the inhibitory properties of CSPGs in SCI, its pharmacokinetics such as enzymatic instability, short half-life, and its potential off target effects in systemic administration has challenged its clinical application (Tester et al., 2007). Pharmacological blockade of LAR and PTP-σ receptors has been successfully achieved through systemic delivery of functionally blocking peptides intracellular LAR peptide (ILP) and intracellular sigma peptide (ISP) in SCI and other neurological conditions (Lang et al., 2015; Hosseini et al., 2022). In our recent work, ILP/ISP co-therapy in conjunction with transplantation of human NPCs showed promising results in promoting NPC-mediated repair in the damaged spinal cord (Hosseini et al., 2022). In these studies, we engrafted directly reprogrammed human caudalized NPCs from adult human bone marrow stromal cells, which hold the potential for autologous transplantation in future clinical applications (Hosseini et al., 2022). Systemic delivery of ILP/ISP through subcutaneous injections starting at the time of NPC transplantation significantly increases long-term survival and biodistribution of engrafted NPCs across the lesion rostro-caudally, which has been a limitation in NPC therapies for SCI (Hosseini et al., 2022). Blocking the CSPG/LAR/PTP-σ axis significantly promotes the differentiation of engrafted NPCs to motoneurons, V1 (EN.1+), and V3 (Sim.1+) interneurons, while it decreases astrogenesis of transplanted NPCs (Hosseini et al., 2022). Importantly, V1 and V3 interneurons are two major populations of spinal interneurons that are predominantly involved in locomotion flexibility, coordination, flexor extensor alteration, and functional recovery following SCI (Hosseini et al., 2022). Transcriptomics revealed that CSPGs inhibit NPCs neurogenesis by deactivating Wnt/β-catenin, a key signaling pathway in central nervous system neurogenesis (Hosseini et al., 2022). ILP/ISP treatment strategy efficiently rescues activation of canonical Wnt signaling by increasing nuclear translocation of the β-catenin complex that leads to restoration of NPC neurogenesis (Hosseini et al., 2022). These studies provided new evidence that SCI-induced upregulation of CSPGs restricts the neurogenic ability of NPCs in the injured spinal cord, at least in part, by blocking Wnt/β-catenin activation (Figure 1). When new NPC-derived neurons are generated, their transition to mature functionally integrated neurons is critical for circuit re-assembly after SCI. This process involves neuronal morphogenesis, neurite elongation, and synapse formation (Fischer et al., 2020). One of the main barriers to the repair process after SCI is the restricted morphoanatomical expansion of neurons in the damaged spinal cord. Dendritic arborization and morphological complexity of spinal neurons are important for neuronal connectivity and transmission of electrical and chemical signals (Alizadeh et al., 2019; Fischer et al., 2020). It is also crucial for newly generated NPC-derived neurons to extend their processes and form synapses with other neurons within the host spinal cord (Hosseini et al., 2024). These studies showed that ILP/ISP treatment restores the inhibitory effects of CSPGs on neurite outgrowth and promotes morphogenesis and maturity of NPC-derived neurons (Hosseini et al., 2022). Of note, the synaptogenesis of NPC-derived neurons is essential for their integration within the host spinal circuitry (Fischer et al., 2020). Interestingly, our in vitro and in vivo experiments indicate that CSPGs impede synaptogenesis of new neurons through activation of LAR and PTP-σ as their co-inhibition enhanced overall synaptogenesis and excitatory synapse formation of transplanted NPC-derived neurons when they are exposed to CSPGs (Hosseini et al., 2022). Of note, LAR and PTP-σ receptors are major presynaptic hubs with significant roles in synapse organization under physiological conditions (Takahashi and Craig, 2013). However, we found improvement in synaptogenesis and spontaneous synaptic transmission of NPC-derived neurons treated with ILP/ISP when exposed to high levels of CSPGs (Hosseini et al., 2022). This may suggest differential roles of LAR and PTP-σ receptors in synapses under physiological and pathological levels of CSPGs. Notably, NPC-derived neurons formed synapses with the host corticospinal tract axons verifying the potential integration of NPC-derived neurons with the supraspinal descending pathways that innervate spinal neurons (Hosseini et al., 2022). In therapeutic applications for SCI, a major goal is achieving improvement in functional recovery. Intriguingly, SCI animals that received transplantation of human NPCs in combination with suppressing CSPG/LAR/PTP-σ axis showed significant improvement in recovery of open field locomotion and sensorimotor integration (Hosseini et al., 2022). Thermal and tactile allodynia and hypersensitivity are two complications after SCI (Alizadeh et al., 2019). Aberrant and increased sprouting of pain afferents is a mechanism underpinning allodynic response after SCI (Karimi-Abdolrezaee et al., 2010). CSPGs are known to control plasticity and their inhibition promotes axonal sprouting (Karimi-Abdolrezaee et al., 2010). To investigate any possible adverse effects of the ISP/ILP treatment strategy on allodynia, we conducted tail flick and Von Frey tests in SCI rats (Hosseini et al., 2022). Our assessment showed that transplantation of human NPCs and/or ILP/ISP treatment does not aggravate thermal and tactile allodynia in rats with chronic SCI (Hosseini et al., 2022). In conclusion, extensive efforts have been made to develop effective cell replacement therapies for SCI. Findings from preclinical studies have shown that the therapeutic benefits of NPC-based therapies are restricted in the dysregulated milieu of SCI. CSPGs have emerged as a negative regulator of NPCs that potently limit the regenerative potential of through receptor-mediated mechanisms. Activation of the CSPG/LAR/PTP-σ axis potently inhibits the multilineage capacity of resident and transplanted NPCs to re-establish the disrupted neuron–glia network after SCI. Pharmacological inhibition of LAR and PTP-σ is a feasible and effective approach to overcome CSPG effects and harness the potential of NPCs in repairing the injured spinal cord. These discoveries have opened new avenues to facilitate the translation of NPC therapies to clinical testing for SCI. SKA acknowledges funding support from the Canadian Institutes of Health Research. SMH was supported by a Doctoral Studentship from the Wings for Life Foundation. C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y

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