Compounds co-targeting kinases in axon regulatory pathways promote regeneration and behavioral recovery after spinal cord injury in mice.

Recovery from spinal cord injury (SCI) and other central nervous system (CNS) trauma is hampered by limits on axonal regeneration in the CNS. Regeneration is restricted by the lack of neuron-intrinsic regenerative capacity and by the repressive microenvironment confronting damaged axons. To address this challenge, we have developed a therapeutic strategy that co-targets kinases involved in both extrinsic and intrinsic regulatory pathways. Prior work identified a kinase inhibitor (RO48) with advantageous polypharmacology (co-inhibition of targets including ROCK2 and S6K1), which promoted CNS axon growth in vitro and corticospinal tract (CST) sprouting in a mouse pyramidotomy model. We now show that RO48 promotes neurite growth from sensory neurons and a variety of CNS neurons in vitro, and promotes CST sprouting and/or regeneration in multiple mouse models of spinal cord injury. Notably, these in vivo effects of RO48 were seen in several independent experimental series performed in distinct laboratories at different times. Finally, in a cervical dorsal hemisection model, RO48 not only promoted growth of CST axons beyond the lesion, but also improved behavioral recovery in the rotarod, gridwalk, and pellet retrieval tasks. Our results provide strong evidence for RO48 as an effective compound to promote axon growth and regeneration. Further, they point to strategies for increasing robustness of interventions in pre-clinical models.

Markerless tracking enables distinction between strategic compensation and functional recovery after spinal cord injury.

Injuries to the cervical spinal cord represent around 60% of all spinal cord injuries (SCIs). A major priority for patients with cervical SCIs is the recovery of any hand or arm function. The similarities between human and rodent "reach-to-eat movements" indicate that analyzing mouse forelimb reaching behavior may be a method of identifying clinically relevant treatments for people with cervical SCIs. One popular behavioral measure of forelimb functional recovery comprises the Single Pellet Retrieval Task (SPRT). The most common outcome measure for this task, however (percentage of pellets successfully retrieved), cannot readily distinguish between recovery of pre-injury motor patterns and strategic compensation. Our objective was to establish outcome measures for the SPRT that are readily adopted by different investigators and capable of measuring recovery of limb function after SCI. We used a simple semi-automated approach to high-speed tracking of mouse forepaw movements during pellet retrieval. DeepLabCut™, a machine learning based computer vision software package, was used to track individual features of the mouse forepaw, allowing a more detailed assessment of reaching behavior after SCI. Interestingly, kinematic analysis of movements pre- and post-injury illuminated persistent deficits in specific features of the reaching motor patterns, namely pronation and paw trajectory, that were poorly correlated with recovery of the ability to successfully retrieve pellets. Thus, we have developed an inexpensive method for detailed analysis of mouse reach-to-eat behavior following SCI. Further, our results suggest that binary success/fail outcome measures primarily assess an animal's ability to compensate rather than a restoration of normal function in the injured pathways and networks.

Developing and evaluating a team development intervention to support interdisciplinary teams.

INTRODUCTION: Incentivizing the development of interdisciplinary scientific teams to address significant societal challenges usually takes the form of pilot funding. However, while pilot funding is likely necessary, it is not sufficient for successful collaborations. Interdisciplinary collaborations are enhanced when team members acquire competencies that support team success. METHODS: We evaluated the impact of a multifaceted team development intervention that included an eight-session workshop spanning two half-days. The workshop employed multiple methods for team development, including lectures on empirically supported best practices, skills-based modules, role plays, hands-on planning sessions, and social interaction within and across teams. We evaluated the impact of the intervention by (1) asking participants to assess each of the workshop sessions and (2) by completing a pre/postquestionnaire that included variables such as readiness to collaborate, goal clarity, process clarity, role ambiguity, and behavioral trust. RESULTS: The content of the team development intervention was very well received, particularly the workshop session focused on psychological safety. Comparison of survey scores before and after the team development intervention indicated that scores on readiness to collaborate and behavioral trust were significantly higher among participants who attended the workshop. Goal clarity, process clarity, and role ambiguity did not differ among those who attended versus those who did not. CONCLUSIONS: Multicomponent team development interventions that focus on key competencies required for interdisciplinary teams can support attitudes and cognitions that the literature on the science of team science indicate are predictive of success. We offer recommendations for the design of future interventions.

Phenotypic Screening Following Transcriptomic Deconvolution to Identify Transcription Factors Mediating Axon Growth Induced by a Kinase Inhibitor.

After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors' combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.

Automation of training and testing motor and related tasks in pre-clinical behavioural and rehabilitative neuroscience.

Testing and training animals in motor and related tasks is a cornerstone of pre-clinical behavioural and rehabilitative neuroscience. Yet manually testing and training animals in these tasks is time consuming and analyses are often subjective. Consequently, there have been many recent advances in automating both the administration and analyses of animal behavioural training and testing. This review is an in-depth appraisal of the history of, and recent developments in, the automation of animal behavioural assays used in neuroscience. We describe the use of common locomotor and non-locomotor tasks used for motor training and testing before and after nervous system injury. This includes a discussion of how these tasks help us to understand the underlying mechanisms of neurological repair and the utility of some tasks for the delivery of rehabilitative training to enhance recovery. We propose two general approaches to automation: automating the physical administration of behavioural tasks (i.e., devices used to facilitate task training, rehabilitative training, and motor testing) and leveraging the use of machine learning in behaviour analysis to generate large volumes of unbiased and comprehensive data. The advantages and disadvantages of automating various motor tasks as well as the limitations of machine learning analyses are examined. In closing, we provide a critical appraisal of the current state of automation in animal behavioural neuroscience and a prospective on some of the advances in machine learning we believe will dramatically enhance the usefulness of these approaches for behavioural neuroscientists.

AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions.

Adeno-associated virus (AAV) is an essential instrument in the neuroscientist's toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8 (AAV8) to environmental pH within the range encountered during endosomal endocytosis (pH 7.4 to pH 4.4), during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between pH 7.4 and pH 6.4, suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the pH-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA (Protocol #18-108-LF) on July 12, 2018.

Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.

Enriched conditioning expands the regenerative ability of sensory neurons after spinal cord injury via neuronal intrinsic redox signaling.

Overcoming the restricted axonal regenerative ability that limits functional repair following a central nervous system injury remains a challenge. Here we report a regenerative paradigm that we call enriched conditioning, which combines environmental enrichment (EE) followed by a conditioning sciatic nerve axotomy that precedes a spinal cord injury (SCI). Enriched conditioning significantly increases the regenerative ability of dorsal root ganglia (DRG) sensory neurons compared to EE or a conditioning injury alone, propelling axon growth well beyond the spinal injury site. Mechanistically, we established that enriched conditioning relies on the unique neuronal intrinsic signaling axis PKC-STAT3-NADPH oxidase 2 (NOX2), enhancing redox signaling as shown by redox proteomics in DRG. Finally, NOX2 conditional deletion or overexpression respectively blocked or phenocopied enriched conditioning-dependent axon regeneration after SCI leading to improved functional recovery. These studies provide a paradigm that drives the regenerative ability of sensory neurons offering a potential redox-dependent regenerative model for mechanistic and therapeutic discoveries.

Computational Identification of Kinases That Control Axon Growth in Mouse.

The determination of signaling pathways and transcriptional networks that control various biological processes is a major challenge from both basic science and translational medicine perspectives. Because such analysis can point to critical disease driver nodes to target for therapeutic purposes, we combined data from phenotypic screening experiments and gene expression studies of mouse neurons to determine information flow through a molecular interaction network using a network propagation approach. We hypothesized that differences in information flow between control and injured conditions prioritize relevant driver nodes that cause this state change. Identifying paths likely taken from potential source nodes to a set of transcription factors (TFs), called sinks, we found that kinases are enriched among source genes sending significantly different amounts of information to TFs in an axonal injury model. Additionally, TFs found to be differentially active during axon growth were enriched in the set of sink genes that received significantly altered amounts of information from source genes. Notably, such enrichment levels hold even when restricting the set of source genes to only those kinases observed to support or hamper neurite growth. That way, we found a set of 71 source genes that send significantly different levels of information to axon growth-relevant TFs. We analyzed their information flow changes in response to axonal injury and their influences on TFs predicted to facilitate or antagonize axon growth. Finally, we drew a network diagram of the interactions and changes in information flow between these source genes and their axon growth-relevant sink TFs.

FAIR SCI Ahead: The Evolution of the Open Data Commons for Pre-Clinical Spinal Cord Injury Research.

Over the last 5 years, multiple stakeholders in the field of spinal cord injury (SCI) research have initiated efforts to promote publications standards and enable sharing of experimental data. In 2016, the National Institutes of Health/National Institute of Neurological Disorders and Stroke hosted representatives from the SCI community to streamline these efforts and discuss the future of data sharing in the field according to the FAIR (Findable, Accessible, Interoperable and Reusable) data stewardship principles. As a next step, a multi-stakeholder group hosted a 2017 symposium in Washington, DC entitled "FAIR SCI Ahead: the Evolution of the Open Data Commons for Spinal Cord Injury research." The goal of this meeting was to receive feedback from the community regarding infrastructure, policies, and organization of a community-governed Open Data Commons (ODC) for pre-clinical SCI research. Here, we summarize the policy outcomes of this meeting and report on progress implementing these policies in the form of a digital ecosystem: the Open Data Commons for Spinal Cord Injury (ODC-SCI.org). ODC-SCI enables data management, harmonization, and controlled sharing of data in a manner consistent with the well-established norms of scholarly publication. Specifically, ODC-SCI is organized around virtual "laboratories" with the ability to share data within each of three distinct data-sharing spaces: within the laboratory, across verified laboratories, or publicly under a creative commons license (CC-BY 4.0) with a digital object identifier that enables data citation. The ODC-SCI implements FAIR data sharing and enables pooled data-driven discovery while crediting the generators of valuable SCI data.

Epigenomic signatures underpin the axonal regenerative ability of dorsal root ganglia sensory neurons.

Axonal injury results in regenerative success or failure, depending on whether the axon lies in the peripheral or the CNS, respectively. The present study addresses whether epigenetic signatures in dorsal root ganglia discriminate between regenerative and non-regenerative axonal injury. Chromatin immunoprecipitation for the histone 3 (H3) post-translational modifications H3K9ac, H3K27ac and H3K27me3; an assay for transposase-accessible chromatin; and RNA sequencing were performed in dorsal root ganglia after sciatic nerve or dorsal column axotomy. Distinct histone acetylation and chromatin accessibility signatures correlated with gene expression after peripheral, but not central, axonal injury. DNA-footprinting analyses revealed new transcriptional regulators associated with regenerative ability. Machine-learning algorithms inferred the direction of most of the gene expression changes. Neuronal conditional deletion of the chromatin remodeler CCCTC-binding factor impaired nerve regeneration, implicating chromatin organization in the regenerative competence. Altogether, the present study offers the first epigenomic map providing insight into the transcriptional response to injury and the differential regenerative ability of sensory neurons.

PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models.

After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.

Scaffold Ranking and Positional Scanning Identify Novel Neurite Outgrowth Promoters with Nanomolar Potency.

Central nervous system (CNS) neurons typically fail to regrow their axons after injury. Injuries or neuropathies that damage CNS axons and disrupt neuronal circuitry often result in permanent functional deficits. Axon regeneration is therefore an intensely pursued therapeutic strategy for numerous CNS disorders. Phenotypic screens utilizing primary neurons have proven successful at identifying agents that promote axon regeneration in vivo. Here, we report the screening of mixture-based combinatorial small molecule libraries in a phenotypic assay utilizing primary CNS neurons and the discovery of neurite outgrowth promoters with low nanomolar potency.

The effect of Jun dimerization on neurite outgrowth and motif binding.

Axon regeneration is a necessary step toward functional recovery after spinal cord injury. The AP-1 transcription factor c-Jun has long been known to play an important role in directing the transcriptional response of Dorsal Root Ganglion (DRG) neurons to peripheral axotomy that results in successful axon regeneration. Here we performed ChIPseq for Jun in mouse DRG neurons after a sciatic nerve crush or sham surgery in order to measure the changes in Jun's DNA binding in response to peripheral axotomy. We found that the majority of Jun's injury-responsive changes in DNA binding occur at putative enhancer elements, rather than proximal to transcription start sites. We also used a series of single polypeptide chain tandem transcription factors to test the effects of different Jun-containing dimers on neurite outgrowth in DRG, cortical and hippocampal neurons. These experiments demonstrated that dimers composed of Jun and Atf3 promoted neurite outgrowth in rat CNS neurons as well as mouse DRG neurons. Our work provides new insight into the mechanisms underlying Jun's role in axon regeneration.

Can Chromatin Accessibility be Exploited for Axon Regeneration?

Several studies have demonstrated that the intrinsic ability of neurons to regenerate their axons can be stimulated by maneuvers that favor the open state of chromatin, such as inhibiting histone deacetylase activity or increasing histone acetyltransferase activity. Taken together, these experiments suggest that axon regenerative ability can be increased by promoting chromatin accessibility. In this article, we assess the direct evidence in the literature for this hypothesis and re-examine other axon regeneration-promoting manipulations to see if they provide additional support. We find that several interventions known to enhance intrinsic axonal growth capability also increase chromatin accessibility. Although the support for this correlation is strong in the literature, we conclude with a word of caution about therapeutics attempting to exploit this relationship.

Publisher Correction: Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

In the version of this Article originally published, the affiliations for Roland A. Fleck and José Antonio Del Río were incorrect due to a technical error that resulted in affiliations 8 and 9 being switched. The correct affiliations are: Roland A. Fleck: 8Centre for Ultrastructural Imaging, Kings College London, London, UK. José Antonio Del Río: 2Cellular and Molecular Neurobiotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain; 9Department of Cell Biology, Physiology and Immunology, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; 10Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain. This has now been amended in all online versions of the Article.

Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-ß1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

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.