Bert Brône

Tear gasses CN, CR, and CS are potent activators of the human TRPA1 receptor

The TRPA1 channel is activated by a number of pungent chemicals, such as allylisothiocyanate, present in mustard oil and thiosulfinates present in garlic. Most of the known activating compounds contain reactive, electrophilic chemical groups, reacting with cysteine residues in the active site of the TRPA1 channel. This covalent modification results in activation of the channel and has been shown to be reversible for several ligands. Commonly used tear gasses CN, CR and CS are also pungent chemicals, and in this study we show that they are extremely potent and selective activators of the human TRPA1 receptor. To our knowledge, these are the most potent TRPA1 agonists known to date. The identification of the molecular target for these tear gasses may open up possibilities to alleviate the effects of tear gasses via treatment with TRPA1 antagonists. In addition these results may contribute to the basic knowledge of the TRPA1 channel that is gaining importance as a pharmacological target.

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time‐lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time‐lapse analysis shows that embryonic microglia are highly dynamic cells.

Analogues of morphanthridine and the tear gas dibenz[b,f][1,4]oxazepine (CR) as extremely potent activators of the human transient receptor potential ankyrin 1 (TRPA1) channel.

The TRPA1 channel can be considered as a key biological sensor to irritant chemicals. In this paper, the discovery of 11H-dibenz[b,e]azepines (morphanthridines) and dibenz[b,f][1,4]oxazepines is described as extremely potent agonists of the TRPA1 receptor. This has led to the discovery that most of the known tear gases are potent TRPA1 activators. The synthesis and biological activity of a number of substituted morphanthridines and dibenz[b,f][1,4]oxazepines have given insight into the SAR around this class of TRPA1 agonists, with EC(50) values ranging from 1 μM to 0.1 nM. Compounds 6 and 32 can be considered as the most potent TRPA1 agonists known to date, with 6 now being used successfully as a screening tool in the discovery of TRPA1 antagonists. The use of ligands such as 6 and 32 as pharmacological tools may contribute to the basic knowledge of the TRPA1 channel and advance the development of TRPA1 antagonists as potential treatment for conditions involving TRPA1 activation, including asthma and pain.

Neurogenic Maturation of Human Dental Pulp Stem Cells Following Neurosphere Generation Induces Morphological and Electrophysiological Characteristics of Functional Neurons

Cell-based therapies are emerging as an alternative treatment option to promote functional recovery in patients suffering from neurological disorders, which are the major cause of death and permanent disability. The present study aimed to differentiate human dental pulp stem cells (hDPSCs) toward functionally active neuronal cells in vitro. hDPSCs were subjected to a two-step protocol. First, neuronal induction was acquired through the formation of neurospheres, followed by neuronal maturation, based on cAMP and neurotrophin-3 (NT-3) signaling. At the ultrastructural level, it was shown that the intra-spheral microenvironment promoted intercellular communication. hDPSCs grew out of the neurospheres in vitro and established a neurogenic differentiated hDPSC culture (d-hDPSCs) upon cAMP and NT-3 signaling. d-hDPSCs were characterized by the increased expression of neuronal markers such as neuronal nuclei, microtubule-associated protein 2, neural cell adhesion molecule, growth-associated protein 43, synapsin I, and synaptophysin compared with nondifferentiated hDPSCs. Enzyme-linked immunosorbent assay demonstrated that the secretion of brain-derived neurotrophic factor, vascular endothelial growth factor, and nerve growth factor differed between d-hDPSCs and hDPSCs. d-hDPSCs acquired neuronal features, including multiple intercommunicating cytoplasmic extensions and increased vesicular transport, as shown by the electron microscopic observation. Patch clamp analysis demonstrated the functional activity of d-hDPSCs by the presence of tetrodotoxin- and tetraethyl ammonium-sensitive voltage-gated sodium and potassium channels, respectively. A subset of d-hDPSCs was able to fire a single action potential. The results reported in this study demonstrate that hDPSCs are capable of neuronal commitment following neurosphere formation, characterized by distinct morphological and electrophysiological properties of functional neuronal cells.

Glycine and glycine receptor signalling in non-neuronal cells

Glycine is an inhibitory neurotransmitter acting mainly in the caudal part of the central nervous system. Besides this neurotransmitter function, glycine has cytoprotective and modulatory effects in different non-neuronal cell types. Modulatory effects were mainly described in immune cells, endothelial cells and macroglial cells, where glycine modulates proliferation, differentiation, migration and cytokine production. Activation of glycine receptors (GlyRs) causes membrane potential changes that in turn modulate calcium flux and downstream effects in these cells. Cytoprotective effects were mainly described in renal cells, hepatocytes and endothelial cells, where glycine protects cells from ischemic cell death. In these cell types, glycine has been suggested to stabilize porous defects that develop in the plasma membranes of ischemic cells, leading to leakage of macromolecules and subsequent cell death. Although there is some evidence linking these effects to the activation of GlyRs, they seem to operate in an entirely different mode from classical neuronal subtypes.

Tricyclic 3,4-dihydropyrimidine-2-thione derivatives as potent TRPA1 antagonists.

The transient receptor potential A1 (TRPA1) channel has been implicated in a number of inflammatory and nociceptive processes, and antagonists of the TRPA1 receptor could offer a potential treatment for conditions such as inflammatory or neuropathic pain, airway disorders, and itch. In a high throughput screen aimed at the identification of TRPA1 antagonists, 4-phenyl-2-thioxo-1,2,3,4-tetrahydro-indeno[1,2-d]pyrimidin-5-one (1) was identified as a potent TRPA1 receptor antagonist. A series of analogous tricyclic 3,4-dihydropyrimidine-2-thiones has been prepared via the multi-component Biginelli reaction and subsequent derivatization. This has led to TRPA1 antagonists with potencies around 10nM for both rat and human derived TRPA1 receptors. The activity was shown to reside exclusively in the 4R-enantiomers.

Maternal immune activation evoked by polyinosinic:polycytidylic acid does not evoke microglial cell activation in the embryo

Several studies have indicated that inflammation during pregnancy increases the risk for the development of neuropsychiatric disorders in the offspring. Morphological brain abnormalities combined with deviations in the inflammatory status of the brain can be observed in patients of both autism and schizophrenia. It was shown that acute infection can induce changes in maternal cytokine levels which in turn are suggested to affect fetal brain development and increase the risk on the development of neuropsychiatric disorders in the offspring. Animal models of maternal immune activation reproduce the etiology of neurodevelopmental disorders such as schizophrenia and autism. In this study the poly (I:C) model was used to mimic viral immune activation in pregnant mice in order to assess the activation status of fetal microglia in these developmental disorders. Because microglia are the resident immune cells of the brain they were expected to be activated due to the inflammatory stimulus. Microglial cell density and activation level in the fetal cortex and hippocampus were determined. Despite the presence of a systemic inflammation in the pregnant mice, there was no significant difference in fetal microglial cell density or immunohistochemically determined activation level between the control and inflammation group. These data indicate that activation of the fetal microglial cells is not likely to be responsible for the inflammation induced deficits in the offspring in this model.

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

Identification of protein networks involved in the disease course of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that warrant further research to study their actual contribution to disease pathology.

Characterization of Na+ currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.