Ribosomal S6 kinases determine intrinsic axonal regeneration capacity.

Why do adult mammalian central nervous system axons not regenerate, when peripheral axons do? Two studies in PLOS Biology point to the role of 2 related ribosomal S6 kinase family members in the differences in regeneration capacity between central and peripheral axons.

An anchor-tether 'hindered' HCN1 inhibitor is antihyperalgesic in a rat spared nerve injury neuropathic pain model.

BACKGROUND: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier. METHODS: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties. RESULTS: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. CONCLUSIONS: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.

Nicotinamide Riboside Improves Enteric Neuropathy in Streptozocin-Induced Diabetic Rats Through Myenteric Plexus Neuroprotection.

BACKGROUND: Diabetes Mellitus causes a systemic oxidative stress due in part to the hyperglycemia and the reactive oxygen species generated. Up to 75% of diabetic patients present with an autonomic neuropathy affecting the Enteric Nervous System. Deficits in the human population are chronic dysmotilities with either increased (i.e., constipation) or decreased (i.e., diarrhea) total gastrointestinal transit times. These are recapitulated in the streptozocin-induced diabetic rat, which is a model of Type I Diabetes Mellitus. AIMS: Examine the effects that a precursor of nicotinamide adenosine dinucleotide (NAD), nicotinamide riboside (NR), had on the development of dysmotility in induced diabetic rats and if fecal microbiota transplant (FMT) could produce the same results. MATERIALS AND METHODS: Utilizing a 6-week treatment paradigm, NR was administered intraperitoneally every 48 h. Total gastrointestinal transit time was assessed weekly utilizing the carmine red method. Three weeks following hyperglycemic induction, FMT was performed between NR-treated animals and untreated animals. SIGNIFICANT RESULTS: There is improvement in overall gastrointestinal transit time with the use of NR. 16S microbiome sequencing demonstrated decreased alpha and beta diversity in induced diabetic rats without change in animals receiving FMT. Improvements in myenteric plexus ganglia density in small and large intestines in diabetic animals treated with NR were seen. CONCLUSIONS: NR treatment led to functional improvement in total gastrointestinal transit time in induced diabetic animals. This was associated with neuroprotection in the myenteric plexuses of both small and large intestines of induced diabetic rats. This represents an important first step in showing NR's benefit as a treatment for diabetic enteric neuropathy. Streptozocin-induced diabetic rats have improved transit times and increased myenteric plexus ganglia density when treated with intraperitoneal nicotinamide riboside.

Semaphorin 3A induces acute changes in membrane excitability in spiral ganglion neurons in vitro.

The development and survival of spiral ganglion neurons (SGNs) are dependent on multiple trophic factors as well as membrane electrical activity. Semaphorins (Sema) constitute a family of membrane-associated and secreted proteins that have garnered significant attention as a potential SGN "navigator" during cochlea development. Previous studies using mutant mice demonstrated that Sema3A plays a role in the SGN pathfinding. The mechanisms, however, by which Sema3A shapes SGNs firing behavior are not known. In these studies, we found that Sema3A plays a novel role in regulating SGN resting membrane potential and excitability. Using dissociated SGN from pre-hearing (P3-P5) and post-hearing mice (P12-P15), we recorded membrane potentials using whole-cell patch clamp recording techniques in apical and basal SGN populations. Recombinant Sema3A was applied to examine the effects on intrinsic membrane properties and action potentials evoked by current injections. Apical and basal SGNs from newborn mice treated with recombinant Sema3A (100 ng/ml) displayed a higher resting membrane potential, higher threshold, decreased amplitude, and prolonged latency and duration of spikes. Although a similar phenomenon was observed in SGNs from post-hearing mice, the resting membrane potential was essentially indistinguishable before and after Sema3A exposure. Sema3A-mediated changes in membrane excitability were associated with a significant decrease in K+ and Ca2+ currents. Sema3A acts through linopirdine-sensitive K+ channels in apical, but not in the basal SGNs. Therefore, Sema3A induces differential effects in SGN membrane excitability that are dependent on age and location, and constitutes an additional early and novel effect of Sema3A SGNs in vitro.

Poly(ADP-ribose) polymerase 1 is a novel target to promote axonal regeneration.

Therapeutic options for the restoration of neurological functions after acute axonal injury are severely limited. In addition to limiting neuronal loss, effective treatments face the challenge of restoring axonal growth within an injury environment where inhibitory molecules from damaged myelin and activated astrocytes act as molecular and physical barriers. Overcoming these barriers to permit axon growth is critical for the development of any repair strategy in the central nervous system. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a previously unidentified and critical mediator of multiple growth-inhibitory signals. We show that exposure of neurons to growth-limiting molecules--such as myelin-derived Nogo and myelin-associated glycoprotein--or reactive astrocyte-produced chondroitin sulfate proteoglycans activates PARP1, resulting in the accumulation of poly(ADP-ribose) in the cell body and axon and limited axonal growth. Accordingly, we find that pharmacological inhibition or genetic loss of PARP1 markedly facilitates axon regeneration over nonpermissive substrates. Together, our findings provide critical insights into the molecular mechanisms of axon growth inhibition and identify PARP1 as an effective target to promote axon regeneration.

Transcriptomic Approaches to Neural Repair.

Understanding why adult CNS neurons fail to regenerate their axons following injury remains a central challenge of neuroscience research. A more complete appreciation of the biological mechanisms shaping the injured nervous system is a crucial prerequisite for the development of robust therapies to promote neural repair. Historically, the identification of regeneration associated signaling pathways has been impeded by the limitations of available genetic and molecular tools. As we progress into an era in which the high-throughput interrogation of gene expression is commonplace and our knowledge base of interactome data is rapidly expanding, we can now begin to assemble a more comprehensive view of the complex biology governing axon regeneration. Here, we highlight current and ongoing work featuring transcriptomic approaches toward the discovery of novel molecular mechanisms that can be manipulated to promote neural repair. SIGNIFICANCE STATEMENT: Transcriptional profiling is a powerful technique with broad applications in the field of neuroscience. Recent advances such as single-cell transcriptomics, CNS cell type-specific and developmental stage-specific expression libraries are rapidly enhancing the power of transcriptomics for neuroscience applications. However, extracting biologically meaningful information from large transcriptomic datasets remains a formidable challenge. This mini-symposium will highlight current work using transcriptomic approaches to identify regulatory networks in the injured nervous system. We will discuss analytical strategies for transcriptomics data, the significance of noncoding RNA networks, and the utility of multiomic data integration. Though the studies featured here specifically focus on neural repair, the approaches highlighted in this mini-symposium will be of broad interest and utility to neuroscientists working in diverse areas of the field.

Axonal transcription factors signal retrogradely in lesioned peripheral nerve.

Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.

cAMP-responsive element-binding protein (CREB) and cAMP co-regulate activator protein 1 (AP1)-dependent regeneration-associated gene expression and neurite growth.

To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration.

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity.

Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of ß-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of ß-actin mRNA, and introducing GFP with the 3'UTR of ß-actin mRNA depletes axons of endogenous ß-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of ß-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

A screen for inducers of p21(waf1/cip1) identifies HIF prolyl hydroxylase inhibitors as neuroprotective agents with antitumor properties.

Preventing neuronal death is a priority for treating neurological diseases. However, therapies that inhibit pathological neuron loss could promote tumorigenesis by preventing the physiological death of cancerous cells. To avert this, we targeted the transcriptional upregulation of p21(waf1/cip1) (p21), an endogenous tumor suppressor with neuroprotective and pro-regenerative activity. We identified potential p21 indcuers by screening a FDA-approved drug and natural product small molecule library against hippocampal HT22 cells stably expressing a luciferase reporter driven by the proximal 60bp of the p21 promoter, and tested them for neuroprotection from glutathione depletion mediated oxidative stress, and cytotoxicity to cancer cell lines (DLD-1, Neuro-2A, SH-SY5Y, NGP, CHLA15, CHP212, and SK-N-SH) in vitro. Of the p21 inducers identified, only ciclopirox, a hypoxia-inducible factor prolyl-4-hydroxylase (HIF-PHD) inhibitor, simultaneously protected neurons from glutathione depletion and decreased cancer cell proliferation at concentrations that were not basally toxic to neurons. We found that other structurally distinct HIF-PHD inhibitors (desferrioxamine, 3,4-dihydroxybenzoate, and dimethyloxalyl glycine) also protected neurons at concentrations that killed cancer cells. HIF-PHD inhibitors stabilize HIF transcription factors, mediating genetic adaptation to hypoxia. While augmenting HIF stability is believed to promote tumorigenesis, we found that chronic HIF-PHD inhibition killed cancer cells, suggesting a protumorigenic role for these enzymes. Moreover, our findings suggest that PHD inhibitors can be used to treat neurological disease without significant concern for cell-autonomous tumor promotion.

Lysophosphatidic acid differentially regulates axonal mRNA translation through 5'UTR elements.

Sensory neurons transport a complex population of mRNAs into their axons, including many encoding ER chaperone proteins. Transport of the mRNA encoding the ER chaperone protein calreticulin is regulated through 3'UTR elements. In other cellular systems, translation of chaperone protein mRNAs can be regulated by ER stress. Here, we have asked if the translation of axonal calreticulin mRNA is regulated in a different manner than its transport into axons. Treatment with lysophosphatidic acid, which is known to trigger axon retraction and stimulate ER Ca(2+) release, caused a translation-dependent increase in axonal calreticulin protein levels. RNA sequences in the 5'UTR of calreticulin confer this translational control through a mechanism that requires an inactivating phosphorylation of eIF2α. In contrast to calreticulin, these signaling events do not activate axonal translation through ß-actin's 5'UTR. Together, these data indicate that stimulation of ER stress can regulate specificity of localized mRNA translation through 5'UTR elements.

Probucol is anti-hyperalgesic in a mouse peripheral nerve injury model of neuropathic pain.

2,6-di-tert-butylphenol (2,6-DTBP) ameliorates mechanical allodynia and thermal hyperalgesia produced by partial sciatic nerve ligation in mice, and selectively inhibits HCN1 channel gating. We hypothesized that the clinically utilized non-anesthetic dimerized congener of 2,6-DTBP, probucol (2,6-di-tert-butyl-4-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol), would relieve the neuropathic phenotype that results from peripheral nerve damage, and that the anti-hyperalgesic efficacy in vivo would correlate with HCN1 channel inhibition in vitro. A single oral dose of probucol (800 mg/kg) relieved mechanical allodynia and thermal hyperalgesia in a mouse spared-nerve injury neuropathic pain model. While the low aqueous solubility of probucol precluded assessment of its possible interaction with HCN1 channels, our results, in conjunction with recent data demonstrating that probucol reduces lipopolysaccharide-induced mechanical allodynia and thermal hyperalgesia, support the testing/development of probucol as a non-opioid, oral antihyperalgesic albeit one of unknown mechanistic action.

Spinal cord injury regulates circular RNA expression in axons.

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

Axonal Localization of transgene mRNA in mature PNS and CNS neurons.

Axonal mRNA transport is robust in cultured neurons but there has been limited evidence for this in vivo. We have used a genetic approach to test for in vivo axonal transport of reporter mRNAs. We show that ß-actin's 3'-UTR can drive axonal localization of GFP mRNA in mature DRG neurons, but mice with γ-actin's 3'-UTR show no axonal GFP mRNA. Peripheral axotomy triggers transport of the ß-actin 3'-UTR containing transgene mRNA into axons. This GFP-3'-ß-actin mRNA accumulates in injured PNS axons before activation of the transgene promoter peaks in the DRG. Spinal cord injury also increases axonal GFP signals in mice carrying this transgene without any increase in transgene expression in the DRGs. These data show for the first time that the ß-actin 3'-UTR is sufficient for axonal localization in both PNS and CNS neurons in vivo.

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs.

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

Regulation of protein levels in subcellular domains through mRNA transport and localized translation.

Localized protein synthesis is increasingly recognized as a means for polarized cells to modulate protein levels in subcellular regions and the distal reaches of their cytoplasm. The axonal and dendritic processes of neurons represent functional domains of cytoplasm that can be separated from their cell body by vast distances. This separation provides a biological setting where the cell uses locally synthesized proteins to both autonomously respond to stimuli and to retrogradely signal the cell body of events occurring is this distal environment. Other cell types undoubtedly take advantage of this localized mechanism, but these have not proven as amenable for isolation of functional subcellular domains. Consequently, neurons have provided an appealing experimental platform for study of mRNA transport and localized protein synthesis. Molecular biology approaches have shown both the population of mRNAs that can localize into axons and dendrites and an unexpectedly complex regulation of their transport into these processes. Several lines of evidence point to similar complexities and specificity for regulation of mRNA translation at subcellular sites. Proteomics studies are beginning to provide a comprehensive view of the protein constituents of subcellular domains in neurons and other cell types. However, these have currently fallen short of dissecting temporal regulation of new protein synthesis in subcellular sites and mechanisms used to ferry mRNAs to these sites.

HDAC6 is a target for protection and regeneration following injury in the nervous system.

Central nervous system (CNS) trauma can result in tissue disruption, neuronal and axonal degeneration, and neurological dysfunction. The limited spontaneous CNS repair in adulthood and aging is often insufficient to overcome disability. Several investigations have demonstrated that targeting HDAC activity can protect neurons and glia and improve outcomes in CNS injury and disease models. However, the enthusiasm for pan-HDAC inhibition in treating neurological conditions is tempered by their toxicity toward a host of CNS cell types -a biological extension of their anticancer properties. Identification of the HDAC isoform, or isoforms, that specifically mediate the beneficial effects of pan-HDAC inhibition could overcome this concern. Here, we show that pan-HDAC inhibition not only promotes neuronal protection against oxidative stress, a common mediator of injury in many neurological conditions, but also promotes neurite growth on myelin-associated glycoprotein and chondroitin sulfate proteoglycan substrates. Real-time PCR revealed a robust and selective increase in HDAC6 expression due to injury in neurons. Accordingly, we have used pharmacological and genetic approaches to demonstrate that inhibition of HDAC6 can promote survival and regeneration of neurons. Consistent with a cytoplasmic localization, the biological effects of HDAC6 inhibition appear transcription-independent. Notably, we find that selective inhibition of HDAC6 avoids cell death associated with pan-HDAC inhibition. Together, these findings define HDAC6 as a potential nontoxic therapeutic target for ameliorating CNS injury characterized by oxidative stress-induced neurodegeneration and insufficient axonal regeneration.

Targeting Affective Mood Disorders With Ketamine to Prevent Chronic Postsurgical Pain.

The phencyclidine-derivative ketamine [2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one] was added to the World Health Organization's Model List of Essential Medicines in 1985 and is also on the Model List of Essential Medicines for Children due to its efficacy and safety as an intravenous anesthetic. In sub-anesthetic doses, ketamine is an effective analgesic for the treatment of acute pain (such as may occur in the perioperative setting). Additionally, ketamine may have efficacy in relieving some forms of chronic pain. In 2019, Janssen Pharmaceuticals received regulatory-approval in both the United States and Europe for use of the S-enantiomer of ketamine in adults living with treatment-resistant major depressive disorder. Pre-existing anxiety/depression and the severity of postoperative pain are risk factors for development of chronic postsurgical pain. An important question is whether short-term administration of ketamine can prevent the conversion of acute postsurgical pain to chronic postsurgical pain. Here, we have reviewed ketamine's effects on the biopsychological processes underlying pain perception and affective mood disorders, focusing on non-NMDA receptor-mediated effects, with an emphasis on results from human trials where available.

G-quadruplex regulation of neural gene expression.

G-quadruplexes are four-stranded helical nucleic acid structures characterized by stacked tetrads of guanosine bases. These structures are widespread throughout mammalian genomic DNA and RNA transcriptomes, and prevalent across all tissues. The role of G-quadruplexes in cancer is well-established, but there has been a growing exploration of these structures in the development and homeostasis of normal tissue. In this review, we focus on the roles of G-quadruplexes in directing gene expression in the nervous system, including the regulation of gene transcription, mRNA processing, and trafficking, as well as protein translation. The role of G-quadruplexes and their molecular interactions in the pathology of neurological diseases is also examined. Outside of cancer, there has been only limited exploration of G-quadruplexes as potential intervention targets to treat disease or injury. We discuss studies that have used small-molecule ligands to manipulate G-quadruplex stability in order to treat disease or direct neural stem/progenitor cell proliferation and differentiation into therapeutically relevant cell types. Understanding the many roles that G-quadruplexes have in the nervous system not only provides critical insight into fundamental molecular mechanisms that control neurological function, but also provides opportunities to identify novel therapeutic targets to treat injury and disease.

A large-scale chemical screen for regulators of the arginase 1 promoter identifies the soy isoflavone daidzeinas a clinically approved small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway.

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.