Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

NK1 antagonists attenuate tau phosphorylation after blast and repeated concussive injury

Exposure to repeated concussive traumatic brain injury (TBI) and to blast-induced TBI has been associated with the potential development of the neurodegenerative condition known as chronic traumatic encephalopathy (CTE). CTE is characterized by the accumulation of hyperphosphorylated tau protein, with the resultant tau tangles thought to initiate the cognitive and behavioral manifestations that appear as the condition progresses. However, the mechanisms linking concussive and blast TBI with tau hyperphosphorylation are unknown. Here we show that single moderate TBI, repeated concussive TBI and blast-induced mild TBI all result in hyperphosphorylation of tau via a substance P mediated mechanism. Post-injury administration of a substance P, NK1 receptor antagonist attenuated the injury-induced phosphorylation of tau by modulating the activity of several key kinases including Akt, ERK1/2 and JNK, and was associated with improvement in neurological outcome. We also demonstrate that inhibition of the TRPV1 mechanoreceptor, which is linked to substance P release, attenuated injury-associated tau hyperphosphorylation, but only when it was administered prior to injury. Our results demonstrate that TBI-mediated stimulation of brain mechanoreceptors is associated with substance P release and consequent tau hyperphosphorylation, with administration of an NK1 receptor antagonist attenuating tau phosphorylation and associated neurological deficits. NK1 antagonists may thus represent a pharmacological approach to attenuate the potential development of CTE following concussive and blast TBI.

Effect of Capsaicinoids on Neurophysiological, Biochemical, and Mechanical Parameters of Swallowing Function

Oropharyngeal dysphagia is prevalent in age-related neurological disorders presenting with impaired efficacy and safety of swallowing due to a loss of muscle force and sensory deficits. Stimulating the oropharynx with capsaicin that mediates Substance P release is an emerging pharmacological treatment option which needs further scientific evidence. Our aim was to comprehensively evaluate the effect of capsaicin on biochemical, neurophysiological, and biomechanical parameters of swallowing function. In a randomized study on healthy individuals, the impact of orally administered capsaicinoids at different dosages and application durations in comparison to non-carbonated water was evaluated. Time course and magnitude of salivary Substance P increase were monitored. Magnetoencephalography was used to detect cortical swallowing network alterations. Modifications in swallowing biomechanics were measured applying high-resolution pharyngeal manometry. Capsaicinoids at 10 μmol/L improved swallowing efficacy as seen by a significant increase of pharyngeal contractile integral and upper esophageal sphincter activation and relaxation times in manometry. Significant improvement of precision in a challenging swallow task accompanied by a reduction in swallowing-related submental electromyographic power was observed with capsaicinoids preconditioning at 10 μmol/L over 5 min, but not with continuous stimulation. The cortical activation pattern remained unchanged after any intervention. A significant increase of salivary Substance P was not detected with 10 μmol/L but with 50 μmol/L and lasted for 15 min after application. Capsaicinoids mediate dose-dependent Substance P release and positively alter swallowing biomechanics in healthy subjects. The results provide supportive evidence for the value of natural capsaicinoids to improve swallowing function.

The mechanism of analgesia in NaV1.7 null mutants

SummaryDeletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain suggesting the locus of analgesia is the nociceptor. Here we demonstrate that NaV1.7 knockout mice have essentially normal nociceptor activity using in vivo calcium imaging and extracellular recording. However, glutamate and substance P release from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also substantially reversed by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid-independent. Male and female humans with NaV1.7 null mutations show naloxone reversible analgesia. Thus opioid-dependent inhibition of neurotransmitter release is the principal mechanism of NaV1.7 null analgesia in mice and humans.

New Engineered-Botulinum Toxins Inhibit the Release of Pain-Related Mediators

Targeted delivery of potent inhibitor of cytokine/pain-mediator into inflammatory or pain-sensing cells is a promising avenue for treating chronic pain, a world-wide major healthcare burden. An unmet need exists for a specific and effective delivery strategy. Herein, we describe a new approach using sortase to site-specifically ligate a non-toxic botulinum neurotoxin D (BoNT/D) core-therapeutic (synaptobrevin-cleaving protease and translocation domains) to cell-specific targeting ligands. An engineered core-therapeutic was efficiently ligated to IL-1β ligand within minutes. The resultant conjugate specifically entered into cultured murine primary macrophages, cleaved synaptobrevin 3 and inhibited LPS/IFN-γ evoked IL-6 release. Likewise, a CGRP receptor antagonist ligand delivered BoNT/D protease into sensory neurons and inhibited K+-evoked substance P release. As cytokines and neuropeptides are major regulators of inflammation and pain, blocking their release by novel engineered inhibitors highlights their therapeutic potential. Our report describes a new and widely-applicable strategy for the production of targeted bio-therapeutics for numerous chronic diseases.

TRPV1 activity and substance P release are required for corneal cold nociception

As a protective mechanism, the cornea is sensitive to noxious stimuli. Here, we show that in mice, a high proportion of corneal TRPM8+ cold-sensing fibers express the heat-sensitive TRPV1 channel. Despite its insensitivity to cold, TRPV1 enhances membrane potential changes and electrical firing of TRPM8+ neurons in response to cold stimulation. This elevated neuronal excitability leads to augmented ocular cold nociception in mice. In a model of dry eye disease, the expression of TRPV1 in TRPM8+ cold-sensing fibers is increased, and results in severe cold allodynia. Overexpression of TRPV1 in TRPM8+ sensory neurons leads to cold allodynia in both corneal and non-corneal tissues without affecting their thermal sensitivity. TRPV1-dependent neuronal sensitization facilitates the release of the neuropeptide substance P from TRPM8+ cold-sensing neurons to signal nociception in response to cold. Our study identifies a mechanism underlying corneal cold nociception and suggests a potential target for the treatment of ocular pain.