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Graphical abstract, Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARβ agonist 4-(5-(4,7-dimethylbenzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (10) with good oral bioavailability in the rat and dog. This molecule increases neurite outgrowth in vitro and induces sensory axon regrowth in vivo in a rodent model of avulsion and crush injury, and thus has the potential for the treatment of nerve injury.

In the CNS, oligodendrocytes are responsible for myelin formation and maintenance. Following spinal cord injury, oligodendrocyte loss and an inhibitory milieu compromise remyelination and recovery. Here, we explored the role of retinoic acid receptor-beta (RARβ) signaling in remyelination. Using a male Sprague Dawley rat model of PNS-CNS injury, we show that oral treatment with a novel drug like RARβ agonist, C286, induces neuronal expression of the proteoglycan decorin and promotes myelination and differentiation of oligodendrocyte precursor cells (NG2+ cells) in a decorin-mediated neuron–glia cross talk. Decorin promoted the activation of RARα in NG2+ cells by increasing the availability of the endogenous ligand RA. NG2+ cells synthesize RA, which is released in association with exosomes. We found that decorin prevents this secretion through regulation of the EGFR–calcium pathway. Using functional and pharmacological studies, we further show that RARα signaling is both required and sufficient for oligodendrocyte differentiation. These findings illustrate that RARβ and RARα are important regulators of oligodendrocyte differentiation, providing new targets for myelination. SIGNIFICANCE STATEMENT This study identifies novel therapeutic targets for remyelination after PNS-CNS injury. Pharmacological and knock-down experiments show that the retinoic acid (RA) signaling promotes differentiation of oligodendrocyte precursor cells (OPCs) and remyelination in a cross talk between neuronal RA receptor-beta (RARβ) and RARα in NG2+ cells. We show that stimulation of RARα is required for the differentiation of OPCs and we describe for the first time how oral treatment with a RARβ agonist (C286, currently being tested in a Phase 1 trial, ISRCTN12424734) leads to the endogenous synthesis of RA through retinaldehyde dehydrogenase 2 (Raldh2) in NG2 cells and controls exosome-associated-RA intracellular levels through a decorin–Ca2+ pathway. Although RARβ has been implicated in distinct aspects of CNS regeneration, this study identifies a novel function for both RARβ and RARα in remyelination.

Stimulation of retinoic acid (RA) mediated signalling pathways following neural injury leads to regeneration in the adult nervous system and numerous studies have shown that the specific activation of the retinoic acid receptor β (RARβ) is required for this process. Here we identify a novel mechanism by which neuronal RARβ activation results in the endogenous synthesis of RA which is released in association with exosomes and acts as a positive cue to axonal/neurite outgrowth. Using an established rodent model of RARβ induced axonal regeneration, we show that neuronal RARβ activation upregulates the enzymes involved in RA synthesis in a cell specific manner; alcohol dehydrogenase7 (ADH7) in neurons and aldehyde dehydrogenase 2 (Raldh2) in NG2 expressing cells (NG2 + cells). These release RA in association with exosomes providing a permissive substrate to neurite outgrowth. Conversely, deletion of Raldh2 in the NG2 + cells in our in vivo regeneration model is sufficient to compromise axonal outgrowth. This hitherto unidentified RA paracrine signalling is required for axonal/neurite outgrowth and is initiated by the activation of neuronal RARβ signalling., Highlights • Raldh2, the enzyme for retinoic acid synthesis, is upregulated in NG2 + cells during axonal regeneration. • Deletion of Raldh2 in NG2 + cells prevents regeneration. • RA signalling modulates axonal pathfinding. • Fine-tuned regulation of RA distribution via exosome transport

Neuronal regeneration is a highly energy-demanding process that greatly relies on axonal mitochondrial transport to meet the enhanced metabolic requirements. Mature neurons typically fail to regenerate after injury, partly because of mitochondrial motility and energy deficits in injured axons. Retinoic acid receptor (RAR)-β signaling is involved in axonal and neurite regeneration. Here we investigate the effect of RAR-β signaling on mitochondria trafficking during neurite outgrowth and find that it enhances their proliferation, speed, and movement toward the growing end of the neuron via hypoxia-inducible factor 1α signaling. We also show that RAR-β signaling promotes the binding of the mitochondria to the anchoring protein, glucose-related protein 75, at the growing tip of neurite, thus allowing them to provide energy and metabolic roles required for neurite outgrowth.—Trigo, D., Goncalves, M. B., Corcoran, J. P. T. The regulation of mitochondria dynamics in neurite outgrowth by retinoic acid receptor β signaling.

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARβ, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARβ (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Failure of axonal regeneration in the central nervous system (CNS) is mainly attributed to a lack of intrinsic neuronal growth programs and an inhibitory environment from a glial scar. Phosphatase and tensin homolog (PTEN) is a major negative regulator of neuronal regeneration and, as such, inhibiting its activity has been considered a therapeutic target for spinal cord (SC) injuries (SCIs). Using a novel model of rat cervical avulsion, we show that treatment with a retinoic acid receptor β (RARβ) agonist results in locomotor and sensory recovery. Axonal regeneration from the severed roots into the SC could be seen by biotinylated dextran amine labeling. Light micrographs of the dorsal root entry zone show the peripheral nervous system (PNS)-CNS transition of regrown axons. RARβ agonist treatment also resulted in the absence of scar formation. Mechanism studies revealed that, inRARβ-agonist-treated neurons,PTENactivity is decreased by cytoplasmic phosphorylation and increased secretion in exosomes. These are taken up by astrocytes, resulting in hampered proliferation and causing them to arrange in a normal-appearing scaffold around the regenerating axons. Attribution of the glial modulation to neuronal PTEN in exosomes was demonstrated by the use of an exosome inhibitor in vivo and PTEN siRNA in vitro assays. The dual effect ofRARβ signaling, both neuronal and neuronal-glial, results in axonal regeneration into the SC after dorsal root neurotmesis. Targeting this pathway may open new avenues for the treatment of SCIs.