Injury distance limits the transcriptional response to spinal injury.

The ability of neurons to sense and respond to damage is fundamental to homeostasis and nervous system repair. For some cell types, notably dorsal root ganglia (DRG) and retinal ganglion cells (RGCs), extensive profiling has revealed a large transcriptional response to axon injury that determines survival and regenerative outcomes. In contrast, the injury response of most supraspinal cell types, whose limited regeneration constrains recovery from spinal injury, is mostly unknown. Here we employed single-nuclei sequencing in mice to profile the transcriptional responses of diverse supraspinal cell types to spinal injury. Surprisingly, thoracic spinal injury triggered only modest changes in gene expression across all populations, including corticospinal tract (CST) neurons. Moreover, CST neurons also responded minimally to cervical injury but much more strongly to intracortical axotomy, including upregulation of numerous regeneration and apoptosis-related transcripts shared with injured DRG and RGC neurons. Thus, the muted response of CST neuron to spinal injury is linked to the injury's distal location, rather than intrinsic cellular characteristics. More broadly, these findings indicate that a central challenge for enhancing regeneration after a spinal injury is the limited sensing of distant injuries and the subsequent modest baseline neuronal response.

Somatostatin regulates central clock function and circadian responses to light.

Daily and annual changes in light are processed by central clock circuits that control the timing of behavior and physiology. The suprachiasmatic nucleus (SCN) in the anterior hypothalamus processes daily photic inputs and encodes changes in day length (i.e., photoperiod), but the SCN circuits that regulate circadian and photoperiodic responses to light remain unclear. Somatostatin (SST) expression in the hypothalamus is modulated by photoperiod, but the role of SST in SCN responses to light has not been examined. Our results indicate that SST signaling regulates daily rhythms in behavior and SCN function in a manner influenced by sex. First, we use cell-fate mapping to provide evidence that SST in the SCN is regulated by light via de novo Sst activation. Next, we demonstrate that Sst  -/- mice display enhanced circadian responses to light, with increased behavioral plasticity to photoperiod, jetlag, and constant light conditions. Notably, lack of Sst  -/- eliminated sex differences in photic responses due to increased plasticity in males, suggesting that SST interacts with clock circuits that process light differently in each sex. Sst  -/- mice also displayed an increase in the number of retinorecipient neurons in the SCN core, which express a type of SST receptor capable of resetting the molecular clock. Last, we show that lack of SST signaling modulates central clock function by influencing SCN photoperiodic encoding, network after-effects, and intercellular synchrony in a sex-specific manner. Collectively, these results provide insight into peptide signaling mechanisms that regulate central clock function and its response to light.

Single Nuclei Analyses Reveal Transcriptional Profiles and Marker Genes for Diverse Supraspinal Populations.

The mammalian brain contains numerous neurons distributed across forebrain, midbrain, and hindbrain that project axons to the lower spinal cord and work in concert to control movement and achieve homeostasis. Extensive work has mapped the anatomic location of supraspinal cell types and continues to establish specific physiological functions. The patterns of gene expression that typify and distinguish these disparate populations, however, are mostly unknown. Here, using adult mice of mixed sex, we combined retrograde labeling of supraspinal cell nuclei with fluorescence-activated nuclei sorting and single-nuclei RNA sequencing analyses to transcriptionally profile neurons that project axons from the brain to lumbar spinal cord. We identified 14 transcriptionally distinct cell types and used a combination of established and newly identified marker genes to assign an anatomic location to each. To validate the putative marker genes, we visualized selected transcripts and confirmed selective expression within lumbar-projecting neurons in discrete supraspinal regions. Finally, we illustrate the potential utility of these data by examining the expression of transcription factors that distinguish different supraspinal cell types and by surveying the expression of receptors for growth and guidance cues that may be present in the spinal cord. Collectively, these data establish transcriptional differences between anatomically defined supraspinal populations, identify a new set of marker genes of use in future experiments, and provide insight into potential differences in cellular and physiological activity across the supraspinal connectome.SIGNIFICANCE STATEMENT The brain communicates with the body through a wide variety of neuronal populations with distinct functions and differential sensitivity to damage and disease. We have used single-nuclei RNA sequencing technology to distinguish patterns of gene expression within a diverse set of neurons that project axons from the mouse brain to the lumbar spinal cord. The results reveal transcriptional differences between populations previously defined on the basis of anatomy, provide new marker genes to facilitate rapid identification of cell type in future work, and suggest distinct responsiveness of different supraspinal populations to external growth and guidance cues.

Brain-wide analysis of the supraspinal connectome reveals anatomical correlates to functional recovery after spinal injury.

The supraspinal connectome is essential for normal behavior and homeostasis and consists of numerous sensory, motor, and autonomic projections from brain to spinal cord. Study of supraspinal control and its restoration after damage has focused mostly on a handful of major populations that carry motor commands, with only limited consideration of dozens more that provide autonomic or crucial motor modulation. Here, we assemble an experimental workflow to rapidly profile the entire supraspinal mesoconnectome in adult mice and disseminate the output in a web-based resource. Optimized viral labeling, 3D imaging, and registration to a mouse digital neuroanatomical atlas assigned tens of thousands of supraspinal neurons to 69 identified regions. We demonstrate the ability of this approach to clarify essential points of topographic mapping between spinal levels, measure population-specific sensitivity to spinal injury, and test the relationships between region-specific neuronal sparing and variability in functional recovery. This work will spur progress by broadening understanding of essential but understudied supraspinal populations.

Widening spinal injury research to consider all supraspinal cell types: Why we must and how we can.

The supraspinal connectome consists of dozens of neuronal populations that project axons from the brain to the spinal cord to influence a wide range of motor, autonomic, and sensory functions. The complexity and wide distribution of supraspinal neurons present significant technical challenges, leading most spinal cord injury research to focus on a handful of major pathways such as the corticospinal, rubrospinal, and raphespinal. Much less is known about many additional populations that carry information to modulate or compensate for these main pathways, or which carry pre-autonomic and other information of high value to individuals with spinal injury. A confluence of technical developments, however, now enables a whole-connectome study of spinal cord injury. Improved viral labeling, tissue clearing, and automated registration to 3D atlases can quantify supraspinal neurons throughout the murine brain, offering a practical means to track responses to injury and treatment on an unprecedented scale. Here we discuss the need for expanded connectome-wide analyses in spinal injury research, illustrate the potential by discussing a new web-based resource for brain-wide study of supraspinal neurons, and highlight future prospects for connectome analyses.

Co-occupancy identifies transcription factor co-operation for axon growth.

Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6 in vitro. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in a mouse model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics approach to detect TF cooperation.

Promotion of corticospinal tract growth by KLF6 requires an injury stimulus and occurs within four weeks of treatment.

Axons in the corticospinal tract (CST) display a limited capacity for compensatory sprouting after partial spinal injuries, potentially limiting functional recovery. Forced expression of a developmentally expressed transcription factor, Krüppel-like factor 6 (KLF6), enhances axon sprouting by adult CST neurons. Here, using a pyramidotomy model of injury in adult mice, we confirm KLF6's pro-sprouting properties in spared corticospinal tract neurons and show that this effect depends on an injury stimulus. In addition, we probed the time course of KLF6-triggered sprouting of CST axons and demonstrate a significant enhancement of growth within four weeks of treatment. Finally, we tested whether KLF6-induced sprouting was accompanied by improvements in forelimb function, either singly or when combined with intensive rehabilitation. We found that regardless of rehabilitative training, and despite robust cross-midline sprouting by corticospinal tract axons, treatment with KLF6 produced no significant improvement in forelimb function on either a modified ladder-crossing task or a pellet-retrieval task. These data clarify important details of KLF6's pro-growth properties and indicate that additional interventions or further optimization will be needed to translate this improvement in axon growth into functional gains.

mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging.

Although ubiquitous in biological studies, the enhanced green and yellow fluorescent proteins (EGFP and EYFP) were not specifically optimized for neuroscience, and their underwhelming brightness and slow expression in brain tissue limits the fidelity of dendritic spine analysis and other indispensable techniques for studying neurodevelopment and plasticity. We hypothesized that EGFP's low solubility in mammalian systems must limit the total fluorescence output of whole cells, and that improving folding efficiency could therefore translate into greater brightness of expressing neurons. By introducing rationally selected combinations of folding-enhancing mutations into GFP templates and screening for brightness and expression rate in human cells, we developed mGreenLantern, a fluorescent protein having up to sixfold greater brightness in cells than EGFP. mGreenLantern illuminates neurons in the mouse brain within 72 h, dramatically reducing lag time between viral transduction and imaging, while its high brightness improves detection of neuronal morphology using widefield, confocal, and two-photon microscopy. When virally expressed to projection neurons in vivo, mGreenLantern fluorescence developed four times faster than EYFP and highlighted long-range processes that were poorly detectable in EYFP-labeled cells. Additionally, mGreenLantern retains strong fluorescence after tissue clearing and expansion microscopy, thereby facilitating superresolution and whole-brain imaging without immunohistochemistry. mGreenLantern can directly replace EGFP/EYFP in diverse systems due to its compatibility with GFP filter sets, recognition by EGFP antibodies, and excellent performance in mouse, human, and bacterial cells. Our screening and rational engineering approach is broadly applicable and suggests that greater potential of fluorescent proteins, including biosensors, could be unlocked using a similar strategy.

Global Connectivity and Function of Descending Spinal Input Revealed by 3D Microscopy and Retrograde Transduction.

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. This complexity presents a major challenge to interpreting functional outcomes from therapies that target supraspinal connectivity after injury or disease, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate that injection of AAV2-Retro to the cervical spinal cord of adult female mice results in highly efficient transduction of supraspinal populations throughout the brainstem, midbrain, and cortex. Some supraspinal populations, including corticospinal and rubrospinal neurons, were transduced with >90% efficiency, with robust transgene expression within 3 d of injection. In contrast, propriospinal and raphe spinal neurons showed much lower rates of retrograde transduction. Using tissue clearing and light-sheet microscopy we present detailed visualizations of descending axons tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors resulted in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde vectors were also highly efficient when injected after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs.SIGNIFICANCE STATEMENT The complexity of descending inputs to the spinal cord presents a major challenge in efforts deliver therapeutics to widespread supraspinal systems, and to interpret their functional effects. Here we demonstrate highly effective gene delivery to diverse supraspinal nuclei using a retrograde viral approach and combine it with tissue clearing and 3D microscopy to map the descending projectome from brain to spinal cord. These data highlight newly developed retrograde viruses as therapeutic and research tools, while offering new insights into supraspinal connectivity.

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 Krüppel-Like Factor Gene Target Dusp14 Regulates Axon Growth and Regeneration.

Purpose: Adult central nervous system (CNS) neurons are unable to regenerate their axons after injury. Krüppel-like transcription factor (KLF) family members regulate intrinsic axon growth ability in vitro and in vivo, but mechanisms downstream of these transcription factors are not known. Methods: Purified retinal ganglion cells (RGCs) were transduced to express exogenous KLF9, KLF16, KLF7, or KLF11; microarray analysis was used to identify downstream genes, which were screened for effects on axon growth. Dual-specificity phosphatase 14 (Dusp14) was further studied using genetic (siRNA, shRNA) and pharmacologic (PTP inhibitor IV) manipulation to assess effects on neurite length in vitro and survival and regeneration in vivo after optic nerve crush in rats and mice. Results: By screening genes regulated by KLFs in RGCs, we identified Dusp14 as a critical gene target limiting axon growth and regeneration downstream of KLF9's ability to suppress axon growth in RGCs. The KLF9-Dusp14 pathway inhibited activation of mitogen-activated protein kinases normally critical to neurotrophic signaling of RGC survival and axon elongation. Decreasing Dusp14 expression or disrupting its function in RGCs increased axon growth in vitro and promoted survival and optic nerve regeneration after optic nerve injury in vivo. Conclusions: These results link intrinsic and extrinsic regulators of axon growth and suggest modulation of the KLF9-Dusp14 pathway as a potential approach to improve regeneration in the adult CNS after injury.

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. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.

High Content Screening of Mammalian Primary Cortical Neurons.

High Content Screening (HCS) can be used to analyze the morphology of neuronal primary cultures on a large scale. When used in the field of neuronal regeneration this approach allows the screening of hundreds or thousands of perturbagens, such as miRNAs, cDNAs, or compounds, for their ability to induce neuronal growth. One of the most important steps while designing these kinds of experiments is the choice of the correct neuronal model. Testing the correct neuronal type is critical to obtain results that are biologically significant and that can later be translated to a clinical setting. For example, if the goal is identifying possible therapies for Spinal Cord Injury (SCI), a challenging target is the neuronal projection from the motor cortex to the spinal cord, the corticospinal tract. Here, we describe the experimental protocols that can be used to produce primary cortical culture from young rat cortices, electroporate the neurons to study the effect of altered gene expression on neurite growth, and immunostain to measure neurite growth parameters.

KLF9 and JNK3 Interact to Suppress Axon Regeneration in the Adult CNS.

Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9's axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9's neurite growth suppression in vitro and promoted axon regeneration in vivo These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS.SIGNIFICANCE STATEMENT Injured CNS nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here, we demonstrate that knocking down Krüppel-like transcription factor 9 (KLF9) via shRNA promotes long-distance axon regeneration after optic nerve injury and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression in vitro and regenerative failure in vivo These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.

The application of CRISPR technology to high content screening in primary neurons.

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editing approach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9 enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in >80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows.

Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury.

Axon regeneration in the central nervous system is limited both by inhibitory extracellular cues and by an intrinsically low capacity for axon growth in some CNS populations. Chondroitin sulfate proteoglycans (CSPGs) are well-studied inhibitors of axon growth in the CNS, and degradation of CSPGs by chondroitinase has been shown to improve the extension of injured axons. Alternatively, axon growth can be improved by targeting the neuron-intrinsic growth capacity through forced expression of regeneration-associated transcription factors. For example, a transcriptionally active chimera of Krüppel-like Factor 7 (KLF7) and a VP16 domain improves axon growth when expressed in corticospinal tract neurons. Here we tested the hypothesis that combined expression of chondroitinase and VP16-KLF7 would lead to further improvements in axon growth after spinal injury. Chondroitinase was expressed by viral transduction of cells in the spinal cord, while VP16-KLF7 was virally expressed in sensory neurons of the dorsal root ganglia or corticospinal tract (CST) neurons. After transection of the dorsal columns, both chondroitinase and VP16-KLF7 increased the proximity of severed sensory axons to the injury site. Similarly, after complete crush injuries, VP16-KLF7 expression increased the approach of CST axons to the injury site. In neither paradigm however, did single or combined treatment with chondroitinase or VP16-KLF7 enable regenerative growth distal to the injury. These results substantiate a role for CSPG inhibition and low KLF7 activity in determining the net retraction of axons from sites of spinal injury, while suggesting that additional factors act to limit a full regenerative response.

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.

Optogenetic Interrogation of Functional Synapse Formation by Corticospinal Tract Axons in the Injured Spinal Cord.

UNLABELLED: To restore function after injury to the CNS, axons must be stimulated to extend into denervated territory and, critically, must form functional synapses with appropriate targets. We showed previously that forced overexpression of the transcription factor Sox11 increases axon growth by corticospinal tract (CST) neurons after spinal injury. However, behavioral outcomes were not improved, raising the question of whether the newly sprouted axons are able to form functional synapses. Here we developed an optogenetic strategy, paired with single-unit extracellular recordings, to assess the ability of Sox11-stimulated CST axons to functionally integrate in the circuitry of the cervical spinal cord. Initial time course experiments established the expression and function of virally expressed Channelrhodopsin (ChR2) in CST cell bodies and in axon terminals in cervical spinal cord. Pyramidotomies were performed in adult mice to deprive the left side of the spinal cord of CST input, and the right CST was treated with adeno-associated virus (AAV)-Sox11 or AAV-EBFP control, along with AAV-ChR2. As expected, Sox11 treatment caused robust midline crossing of CST axons into previously denervated left spinal cord. Clear postsynaptic responses resulted from optogenetic activation of CST terminals, demonstrating the ability of Sox11-stimulated axons to form functional synapses. Mapping of the distribution of CST-evoked spinal activity revealed overall similarity between intact and newly innervated spinal tissue. These data demonstrate the formation of functional synapses by Sox11-stimulated CST axons without significant behavioral benefit, suggesting that new synapses may be mistargeted or otherwise impaired in the ability to coordinate functional output. SIGNIFICANCE STATEMENT: As continued progress is made in promoting the regeneration of CNS axons, questions of synaptic integration are increasingly prominent. Demonstrating direct synaptic integration by regenerated axons and distinguishing its function from indirect relay circuits and target field plasticity have presented technical challenges. Here we force the overexpression of Sox11 to stimulate the growth of corticospinal tract axons in the cervical spinal cord and then use specific optogenetic activation to assess their ability to directly drive postsynaptic activity in spinal cord neurons. By confirming successful synaptic integration, these data illustrate a novel optogenetic-based strategy to monitor and optimize functional reconnection by newly sprouted axons in the injured CNS.

The tumor suppressor HHEX inhibits axon growth when prematurely expressed in developing central nervous system neurons.

Neurons in the embryonic and peripheral nervous system 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.