RARß Agonist Drug (C286) Demonstrates Efficacy in a Pre-clinical Neuropathic Pain Model Restoring Multiple Pathways via DNA Repair Mechanisms.

Neuropathic pain (NP) is associated with profound gene expression alterations within the nociceptive system. DNA mechanisms, such as epigenetic remodeling and repair pathways have been implicated in NP. Here we have used a rat model of peripheral nerve injury to study the effect of a recently developed RARß agonist, C286, currently under clinical research, in NP. A 4-week treatment initiated 2 days after the injury normalized pain sensation. Genome-wide and pathway enrichment analysis showed that multiple mechanisms persistently altered in the spinal cord were restored to preinjury levels by the agonist. Concomitant upregulation of DNA repair proteins, ATM and BRCA1, the latter being required for C286-mediated pain modulation, suggests that early DNA repair may be important to prevent phenotypic epigenetic imprints in NP. Thus, C286 is a promising drug candidate for neuropathic pain and DNA repair mechanisms may be useful therapeutic targets to explore.

Regulation of Myelination by Exosome Associated Retinoic Acid Release from NG2-Positive Cells.

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 (RARß) signaling in remyelination. Using a male Sprague Dawley rat model of PNS-CNS injury, we show that oral treatment with a novel drug like RARß 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 RARß 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 (RARß) 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 RARß 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 RARß has been implicated in distinct aspects of CNS regeneration, this study identifies a novel function for both RARß and RARα in remyelination.

Discovery and lead optimisation of a potent, selective and orally bioavailable RARß agonist for the potential treatment of nerve injury.

Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARß 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.

Amyloid ß inhibits retinoic acid synthesis exacerbating Alzheimer disease pathology which can be attenuated by an retinoic acid receptor α agonist.

The retinoic acid receptor (RAR) α system plays a key role in the adult brain, participating in the homeostatic control of synaptic plasticity, essential for memory function. Here we show that RARα signalling is down-regulated by amyloid beta (Aß), which inhibits the synthesis of the endogenous ligand, retinoic acid (RA). This results in the counteraction of a variety of RARα-activated pathways that are key in the aetiopathology of Alzheimer's disease (AD) but which can be reversed by an RARα agonist. RARα signalling improves cognition in the Tg2576 mice, it has an anti-inflammatory effect and promotes Aß clearance by increasing insulin degrading enzyme and neprilysin activity in both microglia and neurons. In addition, RARα signalling prevents tau phosphorylation. Therefore, stimulation of the RARα signalling pathway using a synthetic agonist, by both clearing Aß and counteracting some of its toxic effects, offers therapeutic potential for the treatment of AD.

Presynaptic efficacy directs normalization of synaptic strength in layer 2/3 rat neocortex after paired activity.

Paired neuronal activity is known to induce changes in synaptic strength that result in the synapse in question having different properties to unmodified synapses. Here we show that in layer 2/3 excitatory connections in young adult rat cortex paired activity acts to normalize the strength and quantal parameters of connections. Paired action potential firing produces long-term potentiation in only a third of connections, whereas a third remain with their amplitude unchanged and a third exhibit long-term depression. Furthermore, the direction of plasticity can be predicted by the initial strength of the connection: weak connections potentiate and strong connections depress. A quantal analysis reveals that changes in synaptic efficacy were predominantly presynaptic in locus and that the key determinant of the direction and magnitude of synaptic modification was the initial release probability (P(r)) of the synapse, which correlated inversely with change in P(r) after pairing. Furthermore, distal synapses also exhibited larger potentiations including postsynaptic increases in efficacy, whereas more proximal inputs did not. This may represent a means by which distal synapses preferentially increase their efficacy to achieve equal weighting at the soma. Paired activity thus acts to normalize synaptic strength, by both pre- and postsynaptic mechanisms.

Detailed passive cable models of layer 2/3 pyramidal cells in rat visual cortex at different temperatures.

We present detailed passive cable models of layer 2/3 pyramidal cells based on somatic voltage transients in response to brief current pulses at physiological and room temperatures and demonstrate how cooling alters the shape of postsynaptic responses. Whole cell recordings were made from cells in visual cortical slices from 20- to 22-day-old rats. The cells were filled with biocytin and morphologies were reconstructed from three cells which were representative of the full range of physiological responses. These formed the basis for electrotonic models with four electrical variables, namely membrane capacitance (C(m)), membrane resistivity (R(m)), cytoplasmic resistivity (R(i)) and a somatic shunt conductance (G(sh)). Simpler models, with a single value for R(m) and no G(sh), did not fit the data adequately. Optimal parameter values were derived by simulating the responses to somatic current pulses, varying the parameters to give the best match to the experimental recordings. G(sh) and R(m) were badly constrained. In contrast, the total membrane conductance (G(tot)) was well constrained, and its reciprocal correlated closely with the slowest membrane time constant (tau(0)). The models showed close agreement for C(m) and R(i) (ranges at 36 degrees C: 0.78-0.94 microF cm(-2) and 140-170 Omegacm), but a larger range for G(tot) (7.2-18.4 nS). Cooling produced consistent effects in all three model cells; C(m) remained constant (Q(10) = 0.96), R(i) increased (Q(10) = 0.80), whilst G(tot) dropped (Q(10) = 1.98). In terms of whole cell physiology, the predominant effect of cooling is to dramatically lengthen the decay of transient voltage shifts. Simulations suggest that this markedly increases the temporal summation of postsynaptic potentials and we demonstrate this effect in the responses of layer 2/3 cells to tetanic extracellular stimulation in layer 4.