Subject(s)
Clinical Trials as Topic , Medical Oncology , Neoplasms/therapy , Precision Medicine , Humans , Neoplasms/geneticsABSTRACT
Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm.
ABSTRACT
BACKGROUND: Ghrelin and obestatin are two gut-derived peptides originating from the same ghrelin/obestatin prepropeptide gene (GHRL). While ghrelin stimulates growth hormone (GH) secretion and food intake and inhibits γ-aminobutyric-acid synaptic transmission onto GHRH (Growth Hormone Releasing Hormone) neurons, obestatin blocks these effects. In Humans, GHRL gene polymorphisms have been associated with pathologies linked to an unbalanced energy homeostasis. We hypothesized that one polymorphism located in the obestatin sequence (Q to L substitution in position 90 of the ghrelin/obestatin prepropeptide, rs4684677) may impact on the function of obestatin. In the present study, we tested the activity of native and Q90L obestatin to modulate ghrelin-induced food intake, GH secretion, cFos activity in GHRH and Neuropeptide Y (NPY) neurons and γ-aminobutyric-acid activity onto GHRH neurons. METHODOLOGY/PRINCIPAL FINDINGS: Food intake, GH secretion and electrophysiological recordings were assessed in C57BL/6 mice. cFos activity was measured in NPY-Renilla-GFP and GHRH-eGFP mice. Mice received saline, ghrelin or ghrelin combined to native or Q90L obestatin (30 nmol each) in the early light phase. Ghrelin stimulation of food intake and GH secretion varied considerably among individual mice with 59-77% eliciting a robust response. In these high-responders, ghrelin-induced food intake and GH secretion were reduced equally by native and Q90L obestatin. In contrast to in vivo observations, Q90L was slightly more efficient than native obestatin in inhibiting ghrelin-induced cFos activation within the hypothalamic arcuate nucleus and the nucleus tractus solitarius of the brainstem. After ghrelin injection, 26% of NPY neurons in the arcuate nucleus expressed cFos protein and this number was significantly reduced by co-administration of Q90L obestatin. Q90L was also more potent that native obestatin in reducing ghrelin-induced inhibition of γ-aminobutyric-acid synaptic transmission onto GHRH neurons. CONCLUSIONS/SIGNIFICANCE: These data support the hypothesis that Q90L obestatin partially blocks ghrelin-induced food intake and GH secretion by acting through NPY and GHRH neurons.
Subject(s)
Feeding Behavior/physiology , Ghrelin/antagonists & inhibitors , Ghrelin/physiology , Growth Hormone-Releasing Hormone/metabolism , Growth Hormone/antagonists & inhibitors , Neurons/metabolism , Neuropeptide Y/metabolism , Animals , Growth Hormone/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BLABSTRACT
Ghrelin, a natural ligand of the growth hormone secretagogue receptor (GHS-R), is synthesized in the stomach but may also be expressed in lesser quantity in the hypothalamus where the GHS-R is located on growth hormone-releasing hormone (GHRH) neurons. Obestatin, a peptide derived from the same precursor as ghrelin, is able to antagonize the ghrelin-induced increase of growth hormone (GH) secretion in vivo but not from pituitary explants in vitro. Thus, the blockade of ghrelin-induced GH release by obestatin could be mediated at the hypothalamic level by the neuronal network that controls pituitary GH secretion. Ghrelin increased GHRH and decreased somatostatin (somatotropin-releasing inhibitory factor) release from hypothalamic explants, whereas obestatin only reduced the ghrelin-induced increase of GHRH release, thus indicating that the effect of ghrelin and obestatin is targeted to GHRH neurons. Patch-clamp recordings on mouse GHRH-enhanced green fluorescent protein neurons indicated that ghrelin and obestatin had no significant effects on glutamatergic synaptic transmission. Ghrelin decreased GABAergic synaptic transmission in 44% of the recorded neurons, an effect blocked in the presence of the GHS-R antagonist BIM28163, and stimulated the firing rate of 78% of GHRH neurons. Obestatin blocked the effects of ghrelin by acting on a receptor different from the GHS-R. These data suggest that: (i) ghrelin increases GHRH neuron excitability by increasing their action potential firing rate and decreasing the strength of GABA inhibitory inputs, thereby leading to an enhanced GHRH release; and (ii) obestatin counteracts ghrelin actions. Such interactions on GHRH neurons probably participate in the control of GH secretion.
Subject(s)
Ghrelin/pharmacology , Growth Hormone-Releasing Hormone/metabolism , Neurons/drug effects , Neurons/metabolism , Synapses/metabolism , Action Potentials/drug effects , Animals , Bicuculline/pharmacology , CHO Cells , Cricetinae , Cricetulus , GABA-A Receptor Antagonists/pharmacology , Ghrelin/metabolism , Glutamic Acid/metabolism , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Patch-Clamp Techniques , Peptide Hormones/pharmacology , Receptors, Ghrelin/metabolism , Somatostatin/metabolism , Synapses/drug effects , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolismABSTRACT
Ghrelin and obestatin are two peptides isolated from the gastrointestinal tract and encoded by the same preproghrelin gene. They convey to the central nervous system informations concerning the nutritional status and/or the energy stores. Ghrelin, mostly acting through the GH secretagogue receptor GHS-R, is a potent GH secretagogue, an orexigenic peptide and a long-term regulator of energy homeostasis. Obestatin was initially described for its anorexigenic effects and its binding to the G protein-coupled receptor 39 (GPR39). However, the role of obestatin is still controversial and the nature of the obestatin receptor remains an open question. This review is focussed on the possible implication of the ghrelin/obestatin system in psychiatric diseases with particular emphasis on eating disorders.
Subject(s)
Body Composition/physiology , Energy Metabolism/physiology , Ghrelin/metabolism , Animals , Feedback, Physiological/physiology , Humans , Receptors, G-Protein-Coupled/metabolism , Receptors, Ghrelin/metabolismABSTRACT
Null mutations in the IL1-receptor accessory protein-like 1 gene (IL1RAPL1) are responsible for an inherited X-linked form of cognitive impairment. IL1RAPL1 protein physically interacts with neuronal calcium sensor-1 (NCS-1), but the functional impact of the IL1RAPL1/NCS-1 interaction remains unknown. Here, we demonstrate that stable expression of IL1RAPL1 in PC12 cells induces a specific silencing of N-type voltage-gated calcium channels (N-VGCC) activity that explains a secretion deficit observed in these IL1RAPL1 cells. Importantly, this modulation of VGCC activity is mediated by NCS-1. Indeed, a specific loss-of-function of N-VGCC was observed in PC12 cells overexpressing NCS-1, and a total recovery of N-VGCC activity was obtained by a down-regulation of NCS-1 in IL1RAPL1 cells. The functional relevance of the interaction between IL1RAPL1 and NCS-1 was also suggested by the reduction of neurite elongation observed in nerve growth factor (NGF)-treated IL1RAPL1 cells, a phenotype rescued by NCS-1 inactivation. Because both proteins are highly expressed in neurons, these results suggest that IL1RAPL1-related mental retardation could result from a disruption of N-VGCC and/or NCS-1-dependent synaptic and neuronal activities.
Subject(s)
Calcium Channels, L-Type/metabolism , Interleukin-1 Receptor Accessory Protein/metabolism , Neurites/metabolism , Animals , Cell Differentiation/drug effects , Electrophysiology , Gene Expression Regulation , Interleukin-1 Receptor Accessory Protein/genetics , Nerve Growth Factor/pharmacology , Neurites/drug effects , Neuronal Calcium-Sensor Proteins/genetics , Neuronal Calcium-Sensor Proteins/metabolism , Neuropeptides/genetics , Neuropeptides/metabolism , PC12 Cells , Patch-Clamp Techniques , RatsABSTRACT
Modulation of glutamatergic transmission by neuropeptides is an essential aspect of neuronal network activity. Activation of the hypothalamic somatostatin sst2 receptor subtype by octreotide decreases AMPA glutamate responses, indicating a central link between a neurohormonal and neuromodulatory peptide and the main hypothalamic fast excitatory neurotransmitter. In mediobasal hypothalamic slices, sst2 activation inhibits the AMPA component of glutamatergic synaptic responses but is ineffective when AMPA currents are pharmacologically isolated. In mediobasal hypothalamic cultures, the decrease of AMPA currents induced by octreotide requires a concomitant activation of sst2 receptors with either NMDA and/or metabotropic glutamate receptors. This modulation depends on changes in intracellular calcium concentration induced by calcium flux through NMDA receptors or calcium release from intracellular stores following metabotropic glutamate receptor activation. These results highlight an unusual regulatory mechanism in which the simultaneous activation of at least three different types of receptor is necessary to allow somatostatin-induced modulation of fast synaptic glutamatergic transmission in the hypothalamus.
Subject(s)
Calcium/metabolism , Hypothalamus, Middle/metabolism , Receptors, AMPA/metabolism , Receptors, Metabotropic Glutamate/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, Somatostatin/metabolism , Animals , Electric Stimulation , Hypothalamus, Middle/physiology , In Vitro Techniques , Intracellular Membranes/metabolism , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Somatostatin/genetics , Synapses/physiologyABSTRACT
Little is known on the influence of epigenetic factors in the developing hypothalamus, a region particularly involved in neuroendocrine regulation and rich in neuropeptides. The present study evaluated the effects of neurotrophins and neuronal activity on neuronal differentiation in hypothalamic cultures sampled from either arcuate or anterior periventricular regions of 17-day-old Sprague-Dawley fetuses. Expression of neuropeptides, tyrosine hydroxylase, neurotrophins and neurotrophin receptors was tested on young (6 days in vitro, DIV) and more mature (14 DIV) cultured neurons by multiple reverse transcription polymerase chain reaction on single cells. In parallel, spontaneous postsynaptic currents were recorded as an index of neuronal connectivity. Neurotrophin-3 (NT3) was expressed in a much larger population of neurons than brain-derived neurotrophic factor (BDNF) at both culture times. At 6 DIV, synaptic currents were scarce and expression of the neurotrophin receptors trkB and trkC was found in a small proportion of neurons only. These parameters increased markedly between 6 and 14 DIV, and also upon addition of neurotrophins. The most striking consequence of arcuate neuron maturation in vitro between 6 and 14 DIV was a marked phenotypic specification affecting somatostatin, neuropeptide Y and pro-opiomelanocortin, the three major neuropeptides expressed in the cultures. NT3, but not BDNF, was able to reproduce maturation-related phenotypic specification in 6 DIV arcuate cultures. Maturation-dependent phenotypic specification was less marked in periventricular cultures; in that case BDNF, not NT3 had a slight effect on phenotype specification. It is concluded that NT3 plays a selective role in phenotypic specification of neuropeptides in the arcuate region, whereas other maturation parameters (neurotrophin receptor expression and/or synaptogenesis) can be potentiated by either neurotrophin in both structures.