Plasticity meets regeneration during innate spinal cord repair

Regenerative capacity of the central nervous system (CNS) is unevenly distributed among vertebrates. While most mammalian species including humans elicit limited repair following CNS injury or disease, highly regenerative vertebrates including urodele amphibians and teleost fish spontaneously reverse CNS damage. Teletost zebrafish (danio rerio) are tropical freshwater fish that proved to be an excellent vertebrate model of successful CNS regeneration. Differential neuronal, glial, and immune injury responses underlie disparate injury outcomes between highly regenerative zebrafish and poorly regenerative mammals. This article describes complications associated with neuronal repair following spinal cord injury (SCI) in poorly regenerative mammals and highlights intersecting modes of plasticity and regeneration in highly regenerative zebrafish (Figures 1 and 2). Comparative approaches evaluating immunoglial SCI responses were recently reviewed elsewhere (Reyes and Mokalled, 2024).

Figure 1.

Neuronal injury responses underlie differential vertebrate regenerative competence after spinal cord injury (SCI).

Schematics denote a neuron-centered representation of neuronal repair mechanisms in zebrafish versus mammals. With the exception of occasional residual sprouting, mammalian spinal cord injury is burdened by neuronal loss, inhibited axon regeneration, and lack of adult neurogenesis. In contrast with mammals, innate spinal cord repair in adult zebrafish is marked by elevated survival and plasticity of surviving neurons, uninhibited axon regeneration, and staggered neurogenesis of excitatory and inhibitory (E/I) neurons.

Regeneration and plasticity in mammalian spinal cord injury: Spinal neurons display a gamut of injury responses that are context-, injury- and species-dependent. For neuron-based regenerative therapies to achieve optimal efficacy, integrating axon regeneration, axon sprouting, and adult neurogenesis represents an ideal strategy that remains to be enacted. Axon regeneration refers to axonal growth from injured neurons (Geoffroy and Zheng, 2014). Despite representing a slow and challenging avenue toward functional recovery, axon regeneration is long-lasting and epitomizes faithful restoration of neural circuitry. Mammalian axon regeneration is challenged by intrinsic neuron inhibition and the need for long-distance regrowth to reconnect distal target sites beyond the lesion. Consequently, long-distance axon regeneration is restricted in the mammalian CNS. Neuronal regeneration or adult neurogenesis denotes de novo regeneration of neuronal cells from adult stem cells. With neurogenesis-based neural repair unattainable in the mammalian spinal cord, stem cell transplantation has been used as an alternative strategy to replenish lost neurons after SCI. Considering the colossal challenges for axonal and neuronal regeneration, neuroplasticity is a third mode of neuronal repair that is thought to be more achievable in mammals. Neuroplasticity refers to the spontaneous reorganization of spared neural circuitry via axon sprouting from uninjured neurons. Plasticity-based rewiring can be more rapid but can only lead to partial, spontaneous recovery. Nonetheless, neuroplasticity does not require long-distance growth, does not rely on de novo cellular or axonal regeneration, and is spontaneously detected after mammalian SCI.

Following SCI, the extent of axon sprouting, axonal and neuronal regeneration dictate the degree of functional recovery (Figure 1). As regeneration-based neural repair is thought to be unattainable in mammals, the mammalian SCI field has directed its efforts to develop plasticity-based repair strategies. For instance, mammalian corticospinal neurons elicit limited axon regeneration and only modest improvement in cellular and functional outcomes using regenerative therapies targeting intrinsic neuron inhibition (Park et al., 2008). Furthermore, neuroplasticity is linked to spontaneous functional recovery following corticospinal injuries (Weidner et al., 2001). Similarly, long descending propriospinal neurons deploy plasticity mechanisms to bypass the injury site, form connections across the lesion, and result in functional recovery within weeks (Courtine et al., 2008). To date, mammalian SCI models, including murine and rat SCI, have been exclusive vertebrate models of neuronal plasticity. Consequently, approaches to study and promote neuroplasticity have been limited to costly, low-throughput testing of candidate molecules or therapies. Expanding the toolset to uncover fundamental mechanisms of neuronal plasticity is a prerequisite to accelerate the development of plasticity-based neural repair strategies.

A new window into neuronal regeneration in adult zebrafish: Unlike mammals, zebrafish reverse paralysis and regain swim function within 6 to 8 weeks of severe SCI (Becker et al., 1997; Burris et al., 2021; Jensen et al., 2023). The advent of single-cell technology allowed for comprehensive temporal understanding of the cell identities that enable spontaneous spinal cord repair (Figure 2). A recently generated single-cell atlas of adult spinal cord repair in zebrafish spanned key regenerative time points – early injury-induced signals at 1 week post-injury (wpi), neuronal and glial regeneration at 3 wpi and cellular remodeling at 6 wpi (Saraswathy et al., 2024). Although SCI triggers multicellular injury responses and increases cell-cell signaling across cell types, bioinformatic assessment of intercellular interactions among spinal cell types uncovered neurons as potent sources of signaling in homeostatic and lesioned SC tissues compared to all other cell types. Injury-induced neuronal signaling pathways include developmental growth factors such as Fibroblast growth factor and WNT, extracellular matrix components such as Fibronectins, and axon guidance molecules such as Semaphorins. Concerted activation of these pathways is instrumental for neuron survival, neurogenesis, axon regrowth, proper axon guidance, and synapse targeting after zebrafish SCI. Mechanistic investigation of these injury-induced signaling pathways will shed further light on specific molecular targets that direct regeneration- or plasticity-driven spinal cord repair.

Figure 2.

Intersecting modes of neuronal plasticity and regeneration in adult zebrafish.

Single-cell transcriptomics of spinal cord regeneration in zebrafish revealed a plasticity signature in a transient population of injury-responsive neurons, or iNeurons. In addition to neuronal plasticity, staggered neurogenesis of excitatory and inhibitory neurons restores E/I balance and contributes to functional recovery after injury. E/I: Excitatory-inhibitory; ERG: ependymo-radial glial cells; iNeuron: injury-responsive neurons; OPC: oligodendrocyte precursor cell; RNA-seq: RNA sequencing; wpi: week post-injury.

Neuronal and axonal regeneration are hallmarks of successful spinal cord repair in adult zebrafish (Figure 1). As the regulatory mechanisms that direct the rate, location, timing, or subtypes of regenerating axons and neurons continue to be unearthed, innate spinal cord repair in zebrafish has evolved from an intriguing, unique biological phenomenon to a valuable SCI discovery platform. Early axon tracing experiments in adult zebrafish showed brainstem neurons are capable of long-distance axon regrowth, extending over 3.5 mm beyond the lesion site (Becker et al., 1997). In addition to axon regrowth, potent populations of adult progenitor cells retaining radial glial features have been shown to replenish lesion-associated neurons. Newly differentiated motor neurons and interneurons populate the regenerate tissue, as pre-existing neurons regrow axons across lesioned tissues. Single-cell transcriptomics and in vivo validation revealed a new appreciation for the regulatory mechanisms that balance the neurogenesis of excitatory and inhibitory neurons after zebrafish SCI (Saraswathy et al., 2024). Altered excitatory-inhibitory (E/I) neurotransmission leads to severe behavioral deficiencies in humans (Isaacson and Scanziani, 2011). Regeneration of appropriate neurons to re-establish E/I balance post-injury is therefore essential to mend CNS function. Temporally, tightly regulated waves of excitatory neuron regeneration precede slower, continuous neurogenesis of inhibitory neurons. Consequently, E/I ratios are marked by early imbalance towards an excitatory phenotype at 1 week post-injury and restoration of baseline E/I activity by 6 weeks post-injury. A detailed understanding of the temporal and molecular identities of spinal progenitors is required to uncover mechanisms that direct sequential neurogenesis of distinct neuron subtypes at different stages of regeneration.

Neuronal plasticity precedes regeneration during innate spinal cord repair: Single-cell transcriptomics uncovered a plasticity-based mode of neuronal repair that precedes regeneration (Figure 2). This recent observation supports a model in which innate spinal cord repair requires more than axon regrowth and de novo neurogenesis to achieve functional recovery. At 3–7 days post-injury, a transient neuronal population referred to as injury-responsive neurons, or iNeurons, acquire a neuroblast-like transcriptional signature. iNeurons are not detected in uninjured spinal cord tissues and their numbers significantly decrease by 3 wpi. In situ hybridization and EdU incorporation were used to determine whether iNeurons represent newly formed regenerating neurons or injury-surviving neurons that support immediate repair following SCI. In these experiments, the majority of iNeurons was EdU negative and possessed an adult, mature neuronal morphology. These in vivo studies indicated iNeurons are injury-surviving neurons that revert to a neuroblast-like state after SCI. Thus, despite their neuroblast-like identities, iNeurons are not newborn. iNeurons express known regeneration-associated genes including atf3 and gap43 and possess an elevated “regeneration score” suggestive of a pro-regenerative role during acute to sub-acute stages of spinal cord repair. Although their exact cell of origin and identities remain to be demonstrated, iNeuron markers including Synaptotagmin XI (syt11b) were shown to co-localize with vglut2a⁺, gad1b⁺, Hb9⁺, isl1⁺, and Pax2⁺ neurons. These observations suggest iNeurons do not represent a specific neuron subtype and that multiple zebrafish neurons survive SCI and elicit an iNeuron transcriptional and regenerative signature during the early stages of spinal cord repair.

The surge of a transient, regeneration-associated signature that precedes axon regrowth and neuronal regeneration raises critical questions regarding the pro-regenerative signature and functions of iNeurons. Tracing experiments using rostral and caudal Biocytin labeling showed iNeurons establish functional connections into the regenerating spinal circuitry as early as 1 wpi. However, finer tracing tools are needed for detailed mapping of iNeuron connectivity. In support of a nodal and instructive role during early recovery from SCI, bioinformatic estimation of cell-cell signaling outgoing from iNeurons and received by other cell types accounted for the majority of all neuronal signaling at 1 wpi. Strong cross-talk between iNeurons and oligodendrocyte precursor cells suggested that reciprocal signaling between these two cell types is a principal feature of spinal cord regeneration. Predicted molecular signaling networks induced by iNeurons include pathways implicated in vesicular trafficking, neuron development, and axon guidance. In a pilot CRISPR/Cas9 genetic screen for genes uniquely expressed in iNeurons, transient mutagenesis of synaptotagmin XI (syt11a/b) and vesicle-associated membrane protein 4 (vamp4) impaired the recovery of swim function following SCI. Syt11 and Vamp4 are vesicle-associated proteins that play different roles in neuronal synapses. Syt11 is a non-canonical SNARE that inhibits spontaneous neurotransmission and bulk endocytosis, whereas Vamp4 promotes Calcium-dependent excitatory neurotransmission release and bulk endocytosis. Consistent with a plasticity-based role after SCI, syt11a/b and vamp4 are not required for cellular regeneration across the lesion, yet crucial for functional recovery post-injury. These findings are consistent with recent mammalian studies showing that inhibition of the presynaptic release machinery is important for axon regrowth (Hilton et al., 2022), and that Syt11 knockout in excitatory forebrain neurons impairs synaptic plasticity and memory (Shimojo et al., 2019). Together, these studies highlight vesicular trafficking as a putatively essential mechanism that underlies spontaneous plasticity and functional recovery after SCI. However, future cell-specific manipulations that enable iNeuron lineage tracing or ablation are needed to better understand and improve neuronal plasticity during spinal cord repair.

The discovery of an acute plasticity signature during innate spinal cord repair unlocks new prospects to leverage the zebrafish model for neuronal plasticity research, in addition to regeneration. In a proof-of-principle genetic screen, a small-scale, single-generation CRISPR/Cas9 screen identified neuronal plasticity genes that are required for spontaneous recovery after zebrafish SCI (Saraswathy et al., 2024). Relative to mammals, zebrafish husbandry is both cost- and space-effective. Consequently, large-scale genetic screening and high-throughput drug screens are readily accessible in zebrafish research. We expect future genetic and pharmacological screens will identify new genetic regulators of neuronal plasticity and will inspire new interventions to promote neuronal survival and plasticity after SCI. To enhance the translational potential of such screens, we propose building a comprehensive screening platform that starts with genetic and/or pharmacological screens in zebrafish and allows for direct and efficient secondary screens in mammalian SCI. Amenable to high throughput genetic and pharmacological studies, zebrafish will be particularly valuable to uncover fundamental mechanisms of neuronal plasticity and to develop new interventions to promote plasticity after SCI.

The emergence of iNeurons after SCI is consistent with a recently identified population of embryonically derived dormant neurons that immediately respond to SCI in larval and adult zebrafish (Vandestadt et al., 2021). Similar to the dormant precursor neurons reported by Vandestadt et al. (2021), more than 90% of iNeurons are not newly generated. Zebrafish iNeurons also share transcriptional similarities with a rare population of mouse spinal neurons that express regeneration associated genes (RAG) including Atf3, Gap43, Sox11, Bdnf, Klf6, Adcyap1, and Syt11 after mouse SCI (Matson et al., 2022). In mice, this rare population of RAG⁺ neurons comprise lumbar spinocerebellar neurons, which elicit axon sparing and undergo structural remodeling including axonal outgrowth after severe contusion injury (Matson et al., 2022). However, as murine RAG⁺ neurons and zebrafish iNeurons downregulate their neuronal markers to upregulate RAGs, further cross-species comparisons and in vivo studies are needed to determine the extent of anatomical and functional similarities between these neuronal populations. We propose that future zebrafish studies will contribute insights and applications into mechanisms of plasticity-driven neural repair.

Summary and perspective: Recovery from spinal cord injury requires intersecting modes of plasticity- and regeneration-based neuronal repair. Until the challenges of axonal and neuronal regeneration get satisfactorily addressed in mammals, promoting plasticity-driven repair will continue to represent an immediate priority in SCI research. Recent zebrafish research uncovered a remarkable neuroplasticity response during acute-to-subacute SCI, highlighting a new avenue to harness the power and high throughput of zebrafish genetics to study neuronal plasticity. Deciphering these mechanisms in zebrafish offers a blueprint to harness and enhance these intrinsic responses for mammalian spinal cord repair.