Glial Bridge Research Articles

Chemical modulation of Akt signaling enhances spinal cord regeneration in zebrafish

Central nervous system lesions often cause permanent motility defects in mammals since the injured neurons cannot regenerate. In contrast, lower vertebrates like zebrafish can regenerate lost neurons and restore motor function. This study investigates the efficacy of SC79, a pan-Akt activator, and A674563, a selective Akt1 inhibitor, as potential therapeutic agents for promoting spinal cord recovery post-injury. Spinal cord injury was induced in zebrafish larvae, and the effects of SC79 and A674563 on neuronal and glial regeneration were examined. SC79 promoted neuronal regeneration without affecting glial bridging, while A674563 induced glial bridging but reduced neuronal regeneration. The combination of SC79 and A674563 induced both glial bridging and neuronal regeneration. Optomotor response tests revealed improved motor function recovery with the combined treatment compared to individual treatments. Additionally, these chemical treatments altered the expression of 12 Akt downstream transcriptional target genes, affirming that the combination treatment preferentially regulates spinal cord regeneration through its action on Akt signaling. These findings highlight the complex interplay of Akt signaling pathways in spinal cord regeneration and suggest potential therapeutic strategies for enhancing functional recovery in spinal cord injury patients

Epigenetic Cross-Talk Between Sirt1 and Dnmt1 Promotes Axonal Regeneration After Spinal Cord Injury in Zebrafish.

Though spinal cord injury (SCI) causes irreversible sensory and motor impairments in human, adult zebrafish retain the potent regenerative capacity by injury-induced proliferation of central nervous system (CNS)-resident progenitor cells to develop new functional neurons at the lesion site. The hallmark of SCI in zebrafish lies in a series of changes in the epigenetic landscape, specifically DNA methylation and histone modifications. Decoding the post-SCI epigenetic modifications is therefore critical for the development of therapeutic remedies that boost SCI recovery process. Here, we have studied on Sirtuin1 (Sirt1), a non-classical histone deacetylase that potentially plays a critical role in neural progenitor cells (NPC) proliferation and axonal regrowth following SCI in zebrafish. We investigated the role of Sirt1 in NPC proliferation and axonal regrowth in response to injury in the regenerating spinal cord and found that Sirt1 is involved in the induction of NPC proliferation along with glial bridging during spinal cord regeneration. We also demonstrate that Sirt1 plays a pivotal role in regulating the HIPPO pathway through deacetylation-mediated inactivation of Dnmt1 and subsequent hypomethylation of yap1 promoter, leading to the induction of ctgfa expression, which drives the NPC proliferation and axonal regrowth to complete the regenerative process. In conclusion, our study reveals a novel cross-talk between two important epigenetic effectors, Sirt1 and Dnmt1, in the context of spinal cord regeneration, establishing a previously undisclosed relation between Sirt1 and Yap1 which provides a deeper understanding of the underlying mechanisms governing injury-induced NPC proliferation and axonal regrowth. Therefore, we have identified Sirt1 as a novel, major epigenetic regulator of spinal cord regeneration by modulating the HIPPO pathway in zebrafish.

Differential Roles of Diet on Development and Spinal Cord Regeneration in Larval Zebrafish.

The zebrafish is a powerful model organism for studying development and regeneration. However, there is a lack of a standardized reference diet for developmental and regeneration experiments. Most studies evaluate the rate of growth, survival, and fecundity. In this study, we compare three diets and their effects on growth and regeneration after a spinal cord injury (SCI). Fish were fed daily for 1 week with daily measurements of overall length and width of spinal injury. Fish fed a live rotifer diet grew 32%, whereas a commercially available diet only led to a 4% increase in body length. Similarly, differences in rate of regeneration were observed with over 80% of rotifer-fed larvae forming a glial bridge after injury compared to <10% of zebrafish fed with the commercial diet. Our data highlight the need for establishing a standardized diet for regeneration studies to improve research reproducibility.

Development and repair of blood vessels in the zebrafish spinal cord.

The vascular system is inefficiently repaired after spinal cord injury (SCI) in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord (SC) and restore motility, but the vascular response to injury has not been investigated. Here, we describe the zebrafish SC blood vasculature, starting in development with the initial vessel ingression in a body size-dependent manner, the acquisition of perivascular support and the establishment of ventral to dorsal blood circulation. The vascular organization grows in complexity and displays multiple barrier specializations in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and axons. Vascular repair involves an early burst of angiogenesis that creates dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-SC barrier is re-established. This study demonstrates that zebrafish can successfully re-vascularize the spinal tissue, reinforcing the value of this organism as a regenerative model for SCI.

Functional trajectories during innate spinal cord repair.

Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Axon growth, glial bridging and adult neurogenesis are hallmarks of cellular regeneration during spinal cord repair. However, the correlation between these cellular regenerative processes and functional recovery remains to be elucidated. Whereas the majority of established functional regeneration metrics measure swim capacity, we hypothesize that gait quality is more directly related to neurological health. Here, we performed a longitudinal swim tracking study for 60 individual zebrafish spanning 8 weeks of spinal cord regeneration. Multiple swim parameters as well as axonal and glial bridging were integrated. We established rostral compensation as a new gait quality metric that highly correlates with functional recovery. Tensor component analysis of longitudinal data supports a correspondence between functional recovery trajectories and neurological outcomes. Moreover, our studies predicted and validated that a subset of functional regeneration parameters measured 1 to 2 weeks post-injury is sufficient to predict the regenerative outcomes of individual animals at 8 weeks post-injury. Our findings established new functional regeneration parameters and generated a comprehensive correlative database between various functional and cellular regeneration outputs.

Progenitor-derived glia are required for spinal cord regeneration in zebrafish.

Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury. Whereas reactive gliosis presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish elicit pro-regenerative bridging functions after injury. Here, we perform genetic lineage tracing, assessment of regulatory sequences and inducible cell ablation to define mechanisms that direct the molecular and cellular responses of glial cells after spinal cord injury in adult zebrafish. Using a newly generated CreERT2 transgenic line, we show that the cells directing expression of the bridging glial marker ctgfa give rise to regenerating glia after injury, with negligible contribution to either neuronal or oligodendrocyte lineages. A 1 kb sequence upstream of the ctgfa gene was sufficient to direct expression in early bridging glia after injury. Finally, ablation of ctgfa-expressing cells using a transgenic nitroreductase strategy impaired glial bridging and recovery of swim behavior after injury. This study identifies key regulatory features, cellular progeny, and requirements of glial cells during innate spinal cord regeneration.

Myostatin is a negative regulator of adult neurogenesis after spinal cord injury in zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

Retinal ganglion cell expression of cytokine enhances occupancy of NG2 cell-derived astrocytes at the nerve injury site: Implication for axon regeneration

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

Transforming growth factor-beta signaling modulates perineurial glial bridging following peripheral spinal motor nerve injury in zebrafish.

Spinal motor nerves are necessary for organismal locomotion and survival. In zebrafish and most vertebrates, these peripheral nervous system structures are composed of bundles of axons that naturally regenerate following injury. However, the cellular and molecular mechanisms that mediate this process are still only partially understood. Perineurial glia, which form a component of the blood‐nerve barrier, are necessary for the earliest regenerative steps by establishing a glial bridge across the injury site as well as phagocytosing debris. Without perineurial glial bridging, regeneration is impaired. In addition to perineurial glia, Schwann cells, the cells that ensheath and myelinate axons within the nerve, are essential for debris clearance and axon guidance. In the absence of Schwann cells, perineurial glia exhibit perturbed bridging, demonstrating that these two cell types communicate during the injury response. While the presence and importance of perineurial glial bridging is known, the molecular mechanisms that underlie this process remain a mystery. Understanding the cellular and molecular interactions that drive perineurial glial bridging is crucial to unlocking the mechanisms underlying successful motor nerve regeneration. Using laser axotomy and in vivo imaging in zebrafish, we show that transforming growth factor‐beta (TGFβ) signaling modulates perineurial glial bridging. Further, we identify connective tissue growth factor‐a (ctgfa) as a downstream effector of TGF‐β signaling that works in a positive feedback loop to mediate perineurial glial bridging. Together, these studies present a new signaling pathway involved in the perineurial glial injury response and further characterize the dynamics of the perineurial glial bridge.

Zebrafish‐inspired strategies for spinal cord repair.

Spinal cord injury (SCI) causes irreversible loss of sensory and motor functions in mammals. In contrast to mammals, adult zebrafish are capable of efficient, spontaneous recovery after SCI. Whereas glial scarring and neuron loss accentuate spinal cord (SC) damage and inhibit regeneration in mammals, zebrafish are capable of pro‐regenerative glial bridging and effective adult neurogenesis post‐injury. Emerging evidence suggests that in mammals these pro‐regenerative processes are either inefficient or overshadowed by anti‐regenerative effects. We propose that developing efficient SCI treatments will require a deeper understanding of endogenous SC regenerative capacity, and that adult zebrafish present a valuable model to uncover fundamental, evolutionarily concealed mechanisms of mammalian SC regeneration.Although glial bridging and neurogenesis are hallmarks of the robust SC repair observed in zebrafish, the mechanisms that direct these processes are underexplored. Using expression profiling and genetic loss‐of‐function, we screened for and identified a suite of molecular factors and cell populations that are activated after injury and required for SC regeneration. These studies revealed that discrete lineage‐restricted niches of progenitor cells differentially contribute to glial bridging and neurogenesis after SCI in zebrafish. Using a battery of genetic and molecular tools, we identified yap‐ctgfa‐twist1a as a central glial bridging signaling axis, generated the first transcriptional profiles of zebrafish bridging glia, and elucidated glial cell injury responses at the single cell level in zebrafish. We are currently using our understanding of the bridging glial cell fate in zebrafish to perform molecular comparisons with mammalian glial cells, and to reprogram mammalian glia into scarless, pro‐regenerative scaffolds that promote SC regrowth. Taken together, our studies elucidate fundamental mechanisms that direct innate SC regeneration in zebrafish, and use natural SC regeneration as a platform to enhance mammalian SC repair at the molecular and cellular levels.Support or Funding InformationWe acknowledge support from NIH (R01‐NS113915), the Missouri Spinal Cord Injury/Disease Research Program, the McDonnell Center for Cellular and Molecular Neuroscience, the Institute of Clinical and Translational Sciences, and from Washington University School of Medicine to M.H.M.

Glucocorticoids Target Ependymal Glia and Inhibit Repair of the Injured Spinal Cord.

Following injury, the mammalian spinal cord forms a glial scar and fails to regenerate. In contrast, vertebrate fish spinal cord tissue regenerates significantly to restore function. Cord transection in zebrafish (Danio rerio) initially causes paralysis and neural cell death. Subsequently, ependymal glia proliferate, bipolar glia extend across the lesion, and new neurons are born; axons from spared and nascent neurons extend along trans-lesional glial bridges to restore functional connectivity. Here we report that glucocorticoids, used in the clinical management of spinal cord injury, directly inhibit neural repair by targeting ependymal glia independently of hematogenous cells and microglia. After transecting injury, the glucocorticoid receptor in ependymal glia is regulated differentially in zebrafish (becoming inactive) vs. the rat (becoming active). Glucocorticoid blockade of neural regeneration via a direct effect on ependymal glia has important therapeutic implications for the putative benefit of corticosteroids in early management of spinal cord injury.

Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration. We performed a genome-wide profiling screen for secreted factors that are up-regulated during zebrafish spinal cord regeneration. We found that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupted spinal cord repair, and transgenic ctgfa overexpression or local delivery of human CTGF recombinant protein accelerated bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration.

Wnt/β-catenin signaling promotes regeneration after adult zebrafish spinal cord injury

Unlike mammals, zebrafish can regenerate their injured spinal cord and regain control of caudal tissues. It was recently shown that Wnt/β-catenin signaling is necessary for spinal cord regeneration in the larval zebrafish. However, the molecular mechanisms of regeneration may or may not be conserved between larval and adult zebrafish. To test this, we assessed the role of Wnt/β-catenin signaling after spinal cord injury in the adult zebrafish. We show that Wnt/β-catenin signaling is increased after spinal cord injury in the adult zebrafish. Moreover, overexpression of Dkk1b inhibited Wnt/β-catenin signaling in the regenerating spinal cord of adult zebrafish. Dkk1b overexpression also inhibited locomotor recovery, axon regeneration, and glial bridge formation in the injured spinal cord. Thus, our data illustrate a conserved role for Wnt/β-catenin signaling in adult and larval zebrafish spinal cord regeneration.

Olfactory ensheathing cells for spinal cord repair: crucial differences between subpopulations of the glia.

OECs for spinal cord repair: Is repairing the injured spinal cord by olfactory ensheathing cell (OEC) transplantation possible? A recent human trial in which a paralysed man regained some function after transplantation of partially purified OECs suggests that this therapy may be a successful approach (Tabakow et al., 2014). In another human trial in which olfactory mucosa lamina propria was transplanted, patients recovered some motor and sensory function (Wang et al., 2015). While these results show promise, it is clear that improvements are needed to provide patients with increased functional output. Strategies to improve the therapeutic use of OECs may include improving the purification of the OECs used for transplantation, using them in combination with growth factors to combat the inhibitory environment and improve axon growth, the use of nerve bridges, advanced physiotherapy and the use of exoskeleton robotics to reinforce functional connections. Of all these approaches, it is probably crucial that the purity of OECs is primarily addressed to ensure consistency in outcomes.

The role of FGF2 in spinal cord trauma and regeneration research

The role of <scp>FGF</scp>2 in spinal cord trauma and regeneration research

Frizzled-3a and Wnt-8b genetically interact during forebrain commissural formation in embryonic zebrafish

The commissural plate forms the rostral surface of the embryonic vertebrate forebrain and provides a cellular substrate for forebrain commissural axons. We have previously reported that the Wnt receptor frizzled-3a (fzd3a) restricts the expression of the chemorepulsive guidance ligand slit2 to a discrete domain of neuroepithelial cells in the commissural plate of embryonic zebrafish. Loss of Fzd3a function perturbed slit2 expression and disrupted the formation of glial bridges which guide the formation of forebrain commissures. We now show that Wnt8b is also necessary for anterior commissural formation as well as for patterning of slit2 expression at the midline. Knock down of Wnt8b produced the same phenotype as loss of Fzd3a which suggested that these genes were acting together to regulate axon guidance. Simultaneous sub-threshold knock down of both Fzd3a and Wnt8b led to a greater than additive increase in the penetrance of the mutant phenotype which indicated that these two genes were indeed interacting. We have shown here that Fzd3a/Wnt8b signaling is essential for normal patterning of the commissural plate and that loss-of-function in either receptor or ligand causes Slit2-dependent defects in glial bridge morphology which indirectly attenuated axon midline crossing in the embryonic vertebrate forebrain.

The Overlap between Headache and Epilepsy in the Light of Recent Advances in Medical Genetics

Migraineurs show an higher frequency of epilepsy (range 1–17%) than general population (0.5–1%), and at the same time migraine prevalence among patients affected by epilepsy is also higher (range 8–15%) than that reported in healthy individuals [11-13]. Especially in children this comorbidity is found often [14]. Seizures and migraine attacks share different pathophysiological mechanisms. Both events may be sign of an underlying brain lesion, or have a probably genetic origin. In this respect, it has been shown that in epilepsy occurs hyperexcitation, while in migraine a brief hyperexcitation period (depolarization) is followed by a long hypoexcitation period (spreading depression), followed again by hyperexcitation, as rebound phenomenon. Moreover, it has been shown that a disexcitability (hyper- and hypo-excitation in the same migraineurs at different points in time) underlines both the phenomenon [1]. Cortical Spreading Depression (CSD) is believed to underlie both migraine and epilepsy [15,16], even in patients affected by migraine without aura. CSD is characterized by a slowly propagating wave of sustained strong neuronal depolarization that generates transient intense spike activity as it progresses into the brain tissue, followed by neural suppression which may last for minutes. The phase of depolarization is associated with a regional cerebral blood flow increase, whereas the phase of reduced neural activity is associated with a reduction in blood flow [17]. It is crucial to remark that while an epileptic discharge can birth exclusively in the cortex, the pathophysiology of headache has its origin at multiple cortico-subcortical levels. Thus, the two cascades of events (headache and epilepsy) show independent ways that can crossover only at the cortical level [18,19]. In headache and epilepsy overlap, after the cortical cascade of events, their onset and propagation are triggered when CSD and epileptic focus reach a definite threshold, lower for CSD than for seizure [6,8,20,21]. Moreover, the onset of CSD and that of the epileptic seizure may facilitate each other [22]. Triggering factors may be environmental or individual (genetically determined or not), determining an ions flow and leading to CSD, through neuronal and glial cytoplasmic bridges rather than through

Fgf-Dependent Glial Cell Bridges Facilitate Spinal Cord Regeneration in Zebrafish

Adult zebrafish show a remarkable capacity to regenerate their spinal column after injury, an ability that stands in stark contrast to the limited repair that occurs within the mammalian CNS post-injury. The reasons for this interspecies difference in regenerative capacity remain unclear. Here we demonstrate a novel role for Fgf signaling during glial cell morphogenesis in promoting axonal regeneration after spinal cord injury. Zebrafish glia are induced by Fgf signaling, to form an elongated bipolar morphology that forms a bridge between the two sides of the resected spinal cord, over which regenerating axons actively migrate. Loss of Fgf function inhibits formation of this "glial bridge" and prevents axon regeneration. Despite the poor potential for mammalian axonal regeneration, primate astrocytes activated by Fgf signaling adopt a similar morphology to that induced in zebrafish glia. This suggests that differential Fgf regulation, rather than intrinsic cell differences, underlie the distinct responses of mammalian and zebrafish glia to injury.

Frizzled-3a and slit2 genetically interact to modulate midline axon crossing in the telencephalon

The anterior commissure forms the first axon connections between the two sides of the embryonic telencephalon. We investigated the role of the transmembrane receptor Frizzled-3a in the development of this commissure using zebrafish as an experimental model. Knock down of Frizzled-3a resulted in complete loss of the anterior commissure. This defect was accompanied by a loss of the glial bridge, expansion of the slit2 expression domain and perturbation of the midline telencephalic–diencephalic boundary. Blocking Slit2 activity following knock down of Frizzled-3a effectively rescued the anterior commissure defect which suggested that Frizzled-3a was indirectly controlling the growth of axons across the rostral midline. We have shown here that Frizzled-3a is essential for normal development of the commissural plate and that loss-of-function causes Slit2-dependent defects in axon midline crossing in the embryonic vertebrate forebrain. These data supports a model whereby Wnt signaling through Frizzled-3a attenuates expression of Slit2 in the rostral midline of the forebrain. The absence of Slit2 facilitates the formation of a midline bridge of glial cells which is used as a substrate for commissural axons. In the absence of this platform of glia, commissural axons fail to cross the rostral midline of the forebrain.

Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re-growth across the gradient. The use of selective enzyme inhibitors revealed that MMP-2 enables immature astrocytes to cross the proteoglycan rim. The bridge-building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pretreatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes coinjected with chondroitinase ABC-induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced.