Transcription factors promote neural regeneration after spinal cord injury.

Abstract

Human spinal cord injury (SCI) results in locomotor and sensory disabilities, which severely affect the quality of life. To restore function after SCI, it is necessary to repair and reconstruct the damaged local circuitry. Major hurdles in neural regeneration include a limited level of neurogenesis in the adult spinal cord and an inflammatory microenvironment that inhibits neurogenesis and axon regeneration. In addition, neurons lost to injury are never replaced. Neural stem/progenitor cells (NSPCs) persist in the adult spinal cord and represent a potential cell source for tissue repair/regeneration after injury, and they are heterogeneous populations with a limited capacity to replenish the lost neuronal population (Hachem et al., 2020; Llorens-Bobadilla et al., 2020). Traumatic injury activates NSPCs in the central nervous system (CNS). However, injury-induced NSPCs largely differentiate into glial cells, i.e., astrocytes and oligodendrocytes, which contribute to glial scar formation. Promoting endogenous NSPCs to differentiate into functional neurons by transcription factors for regeneration and restoration of local neural circuitry is an emerging approach to develop therapeutics for traumatic CNS injury and neurodegenerative disorders. Many transcription factors play critical roles in cell proliferation, cell fate specification, and the generation of specific cell types during the embryonic development of the CNS. Among these factors, genomic screened homeobox 1 (Gsx1 or Gsh1) and NK6 homeobox 1 (Nkx6.1) are known to regulate the proliferation and differentiation of NSPCs (Sander et al., 2000; Pei et al., 2011; Chapman et al., 2018). Gsx1 and Nkx6.1 are genes expressed in NSPCs and intrinsically regulate neurogenesis during the development of the spinal cord. In particular, Gsx1 promotes NSPC maturation and the acquisition of neuronal phenotypes, e.g., the choice between excitatory and inhibitory cell fates of interneurons (Mizuguchi et al., 2006), while Nkx6.1 controls patterning in the formation of the ventral CNS, as well as NSPC specification, migration, and maturation in the neural tube (Sander et al., 2000). We have also established that Gsx1 and Nkx6.1 factors regulate Notch signaling, an essential NSPC signaling pathway, during the embryonic development of the spinal cord and brain (Tzatzalos et al., 2012; Li et al., 2016). The expression level of these transcription factors is low or non-detectable in the adult spinal cord. These findings lead to our hypothesis that reactivation of Gsx1 and Nkx6.1 in the adult injured spinal cord promotes cell proliferation and neural regeneration, and has a therapeutic effect on SCI. To test the efficacy of Gsx1 and Nkx6.1 on neural regeneration and functional recovery after SCI, we examined the roles of the two factors in the adult injured spinal cord using a lentivirus delivery system in a mouse model of lateral hemisection SCI (Patel et al., 2021a, b). Our studies show that lentivirus-mediated expression of Gsx1 or Nkx6.1 promotes NSPC signaling, activates NSPCs, increases cell proliferation and the number of specific interneurons and neuronal activities. It also reduces astrogliosis and glial scar formation at the lesion site (Figure 1). Despite many similar effects of virus-mediated Gsx1 and Nkx6.1 expression on neural regeneration, there is a major difference in functional outcomes. Virus-mediated Gsx1 expression leads to locomotor functional recovery (Patel et al., 2021b), while Nkx6.1 expression does not have a significant benefit in regaining locomotor function (Patel et al., 2021a). This major difference indicates that functional recovery may require the generation of proper cell types and restoration of neuronal connectivity, in addition to reduced glial scar and neuroinflammation.Figure 1: A Venn diagram summarizes the effect of lentivirus-mediated expression of Gsx1 or Nkx6.1 on neural regeneration and locomotor function in a mouse model of lateral hemisection SCI.“↓” Indicates a decrease and “↑” indicates an increase; NSPC: neural stem/progenitor cell; SCI: spinal cord injury.Currently, there are two major approaches in the field that explore neurogenic transcription factors to treat SCI: (i) reprogram residential glial cells into functional neurons; and (ii) promote endogenous NSPCs for neural regeneration. Our studies support that transcription factor-based gene therapy may be a viable treatment for SCI, as we have shown that Gsx1 or Nkx6.1 promote neural regeneration in the adult injured spinal cord. Several other neurogenic transcription factors, e.g., Sox2 and NeuroD1, have been shown to successfully reprogram the resident glial cells into neurons in the injured spinal cord. However, limited or no functional recovery was observed (Su et al., 2014; Puls et al., 2020; Tai et al., 2021). We speculated that the lack of functional recovery in the lineage conversion approach may be due to the following reasons: (i) low reprogramming efficiency, which resulted in an insufficient number of functional neurons; (ii) Sox2-induced neurons resemble GABAergic neurons but with low efficiency (Su et al., 2014), while NeuroD1 converts reactive astrocytes into mostly glutamatergic neurons with high efficiency in the injured spinal cord (Puls et al., 2020). In fact, the induction of additional inhibitory interneurons may contribute to the further imbalance of the excitation/inhibition homeostasis and reducing the excitability of spinal cord inhibitory interneurons could enhance functional recovery in mice with SCI; iii) functional recovery may require the generation of specific cell-types native to the spinal cord. In our study, Gsx1 increases the number of glutamatergic and cholinergic neurons and reduces the number of GABAergic interneurons. These spinal interneurons are essential for transmitting both motor and sensory impulses. It is thus possible that Gsx1-induced functional recovery was partially due to the increased excitatory glutamatergic and cholinergic interneurons and reduced inhibitory GABAergic interneurons. Our findings are also consistent with the established role of Gsx1 to control the generation of excitatory and inhibitory interneurons during embryonic development of the spinal cord (Mizuguchi et al., 2006). At the molecular level, virus-mediated expression of Gsx1 activates resident NSPCs via NSPC signaling pathways, e.g., Notch, Nanog, and Wnt pathways. In acute SCI, Gsx1 expression activates the NSPC-specific signaling pathway and increases proliferation and the number of NSPCs. In chronic SCI, Gsx1 expression increases the population of NSPCs, specific interneurons, serotonergic neuronal activity, and reduces the glial scar, which leads to significant locomotor functional recovery. In comparison, virus-mediated expression of Nkx6.1 also activates resident NSPCs, increases cholinergic neurons, and reduces glial scar formation. It also attenuates neuroinflammation. SCI-induced neuroinflammatory response is beneficial and may be necessary to reduce lesion radius via segregation of the injured tissue. The inflammatory response also promotes phagocytosis of dead and injured cells which is paramount to the regeneration of neural tissue. Despite these important effects of Nkx6.1 at the cellular and molecular levels, Nkx6.1 expression does not lead to functional recovery. Consistent with this observation, virus-mediated expression of Nkx6.1 in mature astrocytes in the cell reprogramming approach fails to convert into functional neurons (Su et al., 2014). The loss of motor and sensory functions after SCI is caused mainly by the severed axon and disrupted neural circuits at the lesion sites. Axon regeneration and synapse formation are thus required to restore the damaged neural circuitry. Axon regeneration in mammals has been a long-standing and extremely challenging problem. Our studies have shown that Gsx1 expression promotes 5-HT neuronal activity, which enhances locomotor functional recovery. 5-HT is an activator of the central pattern generator and is necessary for alternating activity in locomotion. Therefore, small improvements in serotoninergic spinal neurons could lead to a significant increase in locomotor function. In our studies, NSPC populations infected by the Gsx1- or Nkx6.1-encoding viruses likely include Nestin+, NG2+, Foxj1+, and Sox2+ cells. These cells are widely distributed throughout the CNS, display characteristics of NSPCs, and become activated after SCI. After the injury, activated NG2+ cells become primarily glial cells, i.e., oligodendrocytes and astrocytes (Nishiyama et al., 2009). Targeted Sox2 expression unleashes the full neurogenic potential of NG2+ cells, leads to the production of functional neurons, upregulates oligodendrocytes, remyelinates injured axons at the neural lesion, and enhances functional recovery after SCI (Tai et al., 2021). Foxj1+ ependymal cells line the central canal of the spinal cord and lateral ventricles of the brain. These cells are major contributors to the glial scar in the stab SCI model. As a heterogeneous mix of the NSPCs that contribute to neural regeneration after SCI in mammals, targeted approaches to modulate NSPC behavior are necessary for a better understanding of the cellular and molecular mechanism of the transcription factor-based gene therapies. However, it is difficult to target NSPCs because they are not only heterogeneous but also resemble reactive astrocytes in their gene expression profiles, e.g., both cell populations express NSPC markers nestin and vimentin. Currently, it is technically feasible to target NSPCs by selecting AAV serotypes that have high transduction efficiency with NSPCs, e.g., AAV5/6 and AAVrh10, or choosing cell-specific promoters, e.g., NG2 promoter to target NG2+ cells. Future work includes the design and evaluation of targeted gene therapy in NSPCs with neurogenic transcription factors. Specific promoters, e.g., NG2, Sox2, and Foxj1, can be used to direct the expression of transcription factors, e.g., Gsx1 and Nkx6.1, in subpopulations of NSPCs. Studies have shown that targeted Sox2 expression in NG2+ cells in the injured spinal cord contributes to a reduced glial scar and improved locomotor function in contusion, crush, hemisection, and stab models of SCI. Thus, NG2+ cells may represent a viable population for gene therapy to stimulate endogenous neurogenesis in the injured spinal cord. The reactivation of neurogenic transcription factors in the adult may hold the key to successful therapeutic development for traumatic CNS injury and degenerative/demyelinating conditions. Finally, although Nkx6.1 fails to promote locomotor functional recovery, a combination of Gsx1 and Nkx6.1 may provide a better outcome for the treatment of SCI. This work was supported by the State of New Jersey Commission on Spinal Cord Research grant 15IRG006 and Rutgers TechAdvance Fund (to LC). Availability of data and materials:All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional file:Open peer review reports 1 and 2.FigureP-Reviewers: Li H, Labombarda F; C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y.