Gene Regulatory Pathways Driving Central Nervous System Regeneration in Zebrafish

Damage to the central nervous system (CNS) circuitry of adult mammals results in permanent disability. In contrast, the ability to regenerate damaged CNS nerves and achieve functional recovery occurs naturally in fish. The ability of fish to successfully regrow damaged CNS nerves is in part a consequence of their ability to re-express key neuronal growth-associated genes/proteins in response to CNS injury. On such protein is Growth-Associated Protein-43 (Gap43), a protein which is highly enriched in axonal growth cones during CNS development and regeneration. Experiments conducted in mammals have demonstrated that ectopic expression of GAP-43 improves axonal re-growth after injury. Using zebrafish optic nerve as a model for successful CNS regeneration, we have identified that re-expression of the gap43 gene is crucial for regenerative axon growth in vivo. Using a combination of in vivo reporter assays and in vivo regeneration assays, we also identified transcriptional regulatory pathways that are essential for gap43 gene expression in regenerating CNS neurons. We found that transcription factors Ascl1a, Atf3, cJun and Stat3 are required for both re-initiating gap43 expression and driving axon outgrowth in response to optic nerve injury. Futhermore, our results indicate that transcription factors Ascl1a, Atf3 and cJun function cooperatively to re-initiate gap43 expression in a manner that is conserved across highly divergent teleost species. Together, these experiments provide insights into regulatory mechanisms driving successful CNS regeneration, thereby revealing potential targets that may be manipulated to improve regenerative ability in mammals

Selecting Optimal Combinations of Transcription Factors to Promote Axon Regeneration: Why Mechanisms Matter

Recovery from injuries to the central nervous system, including spinal cord injury, is constrained in part by the intrinsically low ability of many CNS neurons to mount an effective regenerative growth response. To improve outcomes, it is essential to understand and ultimately reverse these neuron-intrinsic constraints. Genetic manipulation of key transcription factors (TFs), which act to orchestrate production of multiple regeneration-associated genes, has emerged as a promising strategy. It is likely that no single TF will be sufficient to fully restore neuron-intrinsic growth potential, and that multiple, functionally interacting factors will be needed. An extensive literature, mostly from non-neural cell types, has identified potential mechanisms by which TFs can functionally synergize. Here we examine four potential mechanisms of TF/TF interaction; physical interaction, transcriptional cross-regulation, signaling-based cross regulation, and co-occupancy of regulatory DNA. For each mechanism, we consider how existing knowledge can be used to guide the discovery and effective use of TF combinations in the context of regenerative neuroscience. This mechanistic insight into TF interactions is needed to accelerate the design of effective TF-based interventions to relieve neuron-intrinsic constraints to regeneration and to foster recovery from CNS injury

Epigenetic Profiling Reveals a Developmental Decrease in Promoter Accessibility During Cortical Maturation in vivo

Axon regeneration in adult central nervous system (CNS) is limited in part by a developmental decline in the ability of injured neurons to re-express needed regeneration associated genes (RAGs). Adult CNS neurons may lack appropriate pro-regenerative transcription factors, or may display chromatin structure that restricts transcriptional access to RAGs. Here we performed epigenetic profiling around the promoter regions of key RAGs, and found progressive restriction across a time course of cortical maturation. These data identify a potential intrinsic constraint to axon growth in adult CNS neurons. Neurite outgrowth from cultured postnatal cortical neurons, however, proved insensitive to treatments that improve axon growth in other cell types, including combinatorial overexpression of AP1 factors, overexpression of histone acetyltransferases, and pharmacological inhibitors of histone deacetylases. This insensitivity could be due to intermediate chromatin closure at the time of culture, and highlights important differences in cell culture models used to test potential pro-regenerative interventions

Developmental Chromatin Restriction of Pro‐Growth Gene Networks Acts as an Epigenetic Barrier to Axon Regeneration in Cortical Neurons

Axon regeneration in the central nervous system is prevented in part by a developmental decline in the intrinsic regenerative ability of maturing neurons. This loss of axon growth ability likely reflects widespread changes in gene expression, but the mechanisms that drive this shift remain unclear. Chromatin accessibility has emerged as a key regulatory mechanism in other cellular contexts, raising the possibility that chromatin structure may contribute to the age‐dependent loss of regenerative potential. Here we establish an integrated bioinformatic pipeline that combines analysis of developmentally dynamic gene networks with transcription factor regulation and genome‐wide maps of chromatin accessibility. When applied to the developing cortex, this pipeline detected overall closure of chromatin in sub‐networks of genes associated with axon growth. We next analyzed mature CNS neurons that were supplied with various pro‐regenerative transcription factors. Unlike prior results with SOX11 and KLF7, here we found that neither JUN nor an activated form of STAT3 promoted substantial corticospinal tract regeneration. Correspondingly, chromatin accessibility in JUN or STAT3 target genes was substantially lower than in predicted targets of SOX11 and KLF7. Finally, we used the pipeline to predict pioneer factors that could potentially relieve chromatin constraints at growth‐associated loci. Overall this integrated analysis substantiates the hypothesis that dynamic chromatin accessibility contributes to the developmental decline in axon growth ability and influences the efficacy of pro‐regenerative interventions in the adult, while also pointing toward selected pioneer factors as high‐priority candidates for future combinatorial experiments

KLF6 and STAT3 Co-Occupy Regulatory DNA and Functionally Synergize to Promote Axon Growth in CNS Neurons

The failure of axon regeneration in the CNS limits recovery from damage and disease. Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration, but the underlying mechanisms are unclear. Here we show that forced expression of KLF6 promotes axon regeneration by corticospinal tract neurons in the injured spinal cord. RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in vitro, including sub-networks that were highly enriched for functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis, and bioenergetics. In addition, promoter analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting using ATAC-Seq data confirmed frequent co-occupancy. Co-expression of the two factors yielded a synergistic elevation of neurite growth in vitro. These data clarify the transcriptional control of axon growth and point the way toward novel interventions to promote CNS regeneration

The Tumor Suppressor HHEX Inhibits Axon Growth when Prematurely Expressed in Developing Central Nervous System Neurons

Neurons in the embryonic and peripheral nervoussystem respond to injury by activating transcriptional programs supportive of axon growth, ultimately resulting in functional recovery. In contrast, neurons in the adult central nervous system (CNS) possess a limited capacity to regenerate axons after injury, fundamentally constraining repair. Activating pro-regenerative gene expression in CNS neurons is a promising therapeutic approach, but progress is hampered by incomplete knowledge of the relevant transcription factors. An emerging hypothesis is that factors implicated in cellular growth and motility outside the nervous system may also control axon growth in neurons. We therefore tested sixty-nine transcription factors, previously identified as possessing tumor suppressive or oncogenic properties in non-neuronal cells, in assays of neurite outgrowth. This screen identified YAP1 and E2F1 as enhancers of neurite outgrowth, and PITX1, RBM14, ZBTB16, and HHEX as inhibitors. Follow-up experiments are focused on the tumor suppressor HHEX, one of the strongest growth inhibitors. HHEX is widely expressed in adult CNS neurons, including corticospinal tract neurons after spinal injury, but is present only in trace amounts in immature cortical neurons and adult peripheral neurons. HHEX overexpression in early postnatal cortical neurons reduced both initial axonogenesis and the rate of axon elongation, and domain deletion analysis strongly implicated transcriptional repression as the underlying mechanism. These findings suggest a role for HHEX in restricting axon growth in the developing CNS, and substantiate the hypothesis that previously identified oncogenes and tumor suppressors can play conserved roles in axon extension

MASH1/Ascl1a Leads to GAP43 Expression and Axon Regeneration in the Adult CNS

<div><p>Unlike CNS neurons in adult mammals, neurons in fish and embryonic mammals can regenerate their axons after injury. These divergent regenerative responses are in part mediated by the growth-associated expression of select transcription factors. The basic helix-loop-helix (bHLH) transcription factor, MASH1/Ascl1a, is transiently expressed during the development of many neuronal subtypes and regulates the expression of genes that mediate cell fate determination and differentiation. In the adult zebrafish (Danio rerio), Ascl1a is also transiently expressed in retinal ganglion cells (RGCs) that regenerate axons after optic nerve crush. Utilizing transgenic zebrafish with a 3.6 kb GAP43 promoter that drives expression of an enhanced green fluorescent protein (EGFP), we observed that knock-down of Ascl1a expression reduces both regenerative gap43 gene expression and axonal growth after injury compared to controls. In mammals, the development of noradrenergic brainstem neurons requires MASH1 expression. In contrast to zebrafish RGCs, however, MASH1 is not expressed in the mammalian brainstem after spinal cord injury (SCI). Therefore, we utilized adeno-associated viral (AAV) vectors to overexpress MASH1 in four month old rat (Rattus norvegicus) brainstem neurons in an attempt to promote axon regeneration after SCI. We discovered that after complete transection of the thoracic spinal cord and implantation of a Schwann cell bridge, animals that express MASH1 exhibit increased noradrenergic axon regeneration and improvement in hindlimb joint movements compared to controls. Together these data demonstrate that MASH1/Ascl1a is a fundamental regulator of axonal growth across vertebrates and can induce modifications to the intrinsic state of neurons to promote functional regeneration in response to CNS injury.</p></div

Rats treated with MASH1 exhibit improved hindlimb joint movements.

<p>A, MASH1 treated animals (n = 10) exhibited improved scores over all six weeks on the modified BBB scale compared to controls (n = 11; p<0.05; two-way ANOVA; error bars = SEM). The average scores for control and treated animals at week five were the same, although the SEMs were different. B, At week six, 90% of MASH1 treated animals demonstrated extensive movement of all three hindlimb joints in both legs compared to only 45% of the controls (p<0.05; Fisher’s exact test). Still-frame images from the week 6 BBB test for a control animal (C) and a MASH1 treated animal (D, E): control animal hindlimbs were often flaccid and without movement as depicted in C (arrow) whereas, in contrast, MASH1 treated animals displayed plantar placement and stepping as depicted in D and E (arrows).</p

Rats treated with MASH1 exhibit increased regeneration of noradrenergic axons into the SC bridge.

<p>A, A low magnification image of a SC bridge from a MASH1 treated animal showing the rostral (left) and caudal (right) spinal cord/SC bridge interfaces delineated by GFAP-positive astrocytes (white). Note the long astrocyte processes (small arrowheads, A and B) that extend into the bridge. Hoechst-stained nuclei are blue and the large arrowhead indicates the polymer channel. Small holes (arrows) in the top of the channel were created to inject a fluid mixture of SCs and Matrigel (scale bar = 1 mm). Locations of higher magnification images taken on an adjacent section are indicated by letters (B-G). In treated animals, many beaded DβH-positive axons (red, arrows) regenerated 0.25 mm (B), 0.5 mm (C), 1.0 mm (D), 1.5 mm (E), 2.0 mm (F), and more than 2.5 mm (G), beyond the rostral spinal cord/SC bridge interface (scale bar = 20 μm). H, An illustration of the line-transect method of analysis, depicting DβH-positive axons (red) regenerating into a SC bridge. The polymer channel (thick black lines) and the transverse dorso-ventral lines used for quantification (thin purple lines) are diagrammed. The numbers represent mm from the rostral interface. I, The percentage of DβH-positive axons 10 mm rostral to the bridge that regenerated across the bridge was greater in MASH1 treated animals (n = 10) compared to control animals (n = 11; ** = p<0.01, two-way ANOVA; ++ = p<0.01 Bonferroni posttest).</p

Ascl1a is required for RGC axon regeneration in zebrafish.

<p>A-L, Cross sections through the retina focused on the retinal ganglion cell layer. Four days after optic nerve transection, zebrafish RGCs that received MOs (red, arrowheads) were retrogradely traced with dextran (green) 1 mm beyond the site of injury. Animals that received negative control MOs (A-D) were able to regenerate RGC axons as evident by the co-localization of MOs and tracer (yellow, arrow). In contrast few RGCs regenerated axons in animals that received MOs to knockdown GAP43 (E-H) or Ascl1a (I-L). DAPI stained nuclei are blue (scale bar = 10 μm). M, Quantification of the percentage of RGCs that received the MOs and were able to regenerate axons. Compared to controls (n = 6), those that received either GAP43 (n = 6) or Ascl1a (n = 6) MOs exhibited a reduced percentage of RGC axons to regenerate (*** = p<0.001 one-way ANOVA; +++ = p<0.001 Bonferroni posttest). Compared to animals that received GAP43 MOs, those that received Ascl1a MOs exhibited a greater reduction in the percentage of RGC axons to regenerate (*** = p<0.001, t-test).</p