Generation of iPSC-Derived Human Peripheral Sensory Neurons Releasing Substance P Elicited by TRPV1 Agonists.

Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro.

The prion protein selectively binds to and modulates the content of purinergic receptor P2X4R.

The GPI-anchored prion protein (PrP(C)) is involved in neurodegeneration, either through misfolding in the Transmissible Spongiform Encephalopathies (TSE), or as a mediator of the neurotoxicity of peptide oligomers in Alzheimer's Disease. PrP(C) has been attributed pleiotropic functions, and appears to scaffold a variety of cell surface signaling modules, for example through its binding to several neurotransmitter receptors. Here we used transfected HEK293 cells to test for an interaction of PrP(C) with purinergic receptor P2X4R. The prion protein bound P2X4R in both overlay and co-immunoprecipitation assays, and co-localized mostly intracellularly, but occasionaly at the cell surface in confocal micrographs. Functional PrP(C):P2X4R interaction was tested by the uptake of a P2X4R-permeant compound, and by modulation of intracellular calcium. Unexpectedly, however, this interaction was traced to a selective effect of PrP(C) upon the content of co-transfected P2X4R. The results suggest a role of PrP(C) in proteostasis, dysfunctions of which may be involved in the pathogenesis of neurodegenerative diseases such as TSE and Alzheimer's Disease.

LASSBio-1135: a dual TRPV1 antagonist and anti-TNF-alpha compound orally effective in models of inflammatory and neuropathic pain.

LASSBio-1135 is an imidazo[1,2-a]pyridine derivative with high efficacy in screening models of nociception and inflammation, presumed as a weak COX-2 inhibitor. In order to tease out its mechanism of action, we investigated others possible target for LASSBio-1135, such as TNF-α and TRPV1, to better characterize it as a multitarget compound useful in the treatment of chronic pain. TRPV1 modulation was assessed in TRPV1-expressing Xenopus oocytes against capsaicin and low pH-induced current. Modulation of TNF-α production was evaluated in culture of macrophages stimulated with LPS. In vivo efficacy of LASSBio-1135 was investigated in carrageenan and partial sciatic ligation-induced thermal hyperalgesia and mechanical allodynia. Corroborating its previous demonstration of efficacy in a model of capsaicin-induced hyperalgesia, LASSBio-1135 blocks capsaicin-elicited currents in a non-competitive way with an IC50 of 580 nM as well as low pH-induced current at 50 µM. As an additional action, LASSBio-1135 inhibited TNF-α release in these cells stimulated by LPS with an IC50 of 546 nM by reducing p38 MAPK phosphorilation. Oral administration of 100 µmol x Kg(-1) LASSBio-1135 markedly reduced thermal hyperalgesia induced by carrageenan, however at 10 µmol x Kg(-1) only a partial reduction was observed at the 4th h. Neutrophil recruitment and TNF-α production after carrageenan stimulus was also inhibited by the treatment with LASSBio-1135. Modulating TRPV1 and TNF-α production, two key therapeutic targets of neuropathic pain, 100 µmol x Kg(-1) LASSBio-1135 was orally efficacious in reversing thermal hyperalgesia and mechanical allodynia produced by partial sciatic ligation 7-11 days after surgery without provoking hyperthermia, a common side effect of TRPV1 antagonists. In conclusion LASSBio-1135, besides being a weak COX-2 inhibitor, is a non-competitive TRPV1 antagonist and a TNF-α inhibitor. As a multitarget compound, LASSBio-1135 is orally efficacious in a model of neuropathic pain without presenting hyperthermia.

Erythrina mulungu alkaloids are potent inhibitors of neuronal nicotinic receptor currents in mammalian cells.

Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4ß2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4ß2 nicotinic acetylcholine receptors. The IC50 obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4ß2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4ß2 subtype.

Anti-inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabinoid receptor.

Allosteric modulation of G-protein-coupled receptors represents a key goal of current pharmacology. In particular, endogenous allosteric modulators might represent important targets of interventions aimed at maximizing therapeutic efficacy and reducing side effects of drugs. Here we show that the anti-inflammatory lipid lipoxin A(4) is an endogenous allosteric enhancer of the CB(1) cannabinoid receptor. Lipoxin A(4) was detected in brain tissues, did not compete for the orthosteric binding site of the CB(1) receptor (vs. (3)H-SR141716A), and did not alter endocannabinoid metabolism (as opposed to URB597 and MAFP), but it enhanced affinity of anandamide at the CB1 receptor, thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition, lipoxin A(4) displayed a CB(1) receptor-dependent protective effect against ß-amyloid (1-40)-induced spatial memory impairment in mice. The discovery of lipoxins as a class of endogenous allosteric modulators of CB(1) receptors may foster the therapeutic exploitation of the endocannabinoid system, in particular for the treatment of neurodegenerative disorders.

Cannabinoids modulate cell survival in embryoid bodies.

ESCs (embryonic stem cells) are potentially able to replace damaged cells in animal models of neural pathologies such as Parkinson's disease, stroke and spinal cord lesions. Nevertheless, many issues remain unsolved regarding optimal culturing procedures for these cells. For instance, on their path to differentiation in vitro, which usually involves the formation of EBs (embryoid bodies), they may present chromosomal instability, loss of pluripotency or simply die. Therefore, finding strategies to increase the survival of cells within EBs is of great interest. Cannabinoid receptors have many roles in the physiology of the adult body, but little is known about their role in the biology of ESCs. Herein, we investigated how two cannabinoid receptors, CB1 and CB2, may affect the outcome of ESCs aggregated as EBs. RT-PCR (reverse transcriptase-PCR) revealed that EBs expressed both CB1 and CB2 receptors. Aggregation of ESCs into EBs followed by 2-day incubation with a CB1/CB2 agonist reduced cell death by approximately 45%, which was reversed by a CB1 antagonist. A specific CB2 agonist also reduced cell death by approximately 20%. These data indicate that both cannabinoid receptors, CB1 and CB2, are involved in reducing cell death in EBs mediated by exogenous cannabinoids. No increase in proliferation, neural differentiation or changes in chromosomal stability was observed. This study indicates that cannabinoid signalling is functionally implicated in the biology of differentiating ESCs, being the first to show that activation of cannabinoid receptors is able to increase cell viability via reduction of cell death rate in EBs.

Isoform specificity of P2X2 purinergic receptor C-terminus binding to tubulin.

Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X2 is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X2 binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X2 with a naturally occurring splicing variant named P2X2b. These isoforms differ in their C-terminal regions which contain the proposed beta-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X2 binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X2 C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X2 interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles.