Attenuation of fibroblast activation and fibrosis by adropin in systemic sclerosis.

Fibrotic diseases impose a major socioeconomic challenge on modern societies and have limited treatment options. Adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize adropin as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent down-regulation of adropin/ENHO in skin across multiple cohorts of patients with SSc. The prototypical profibrotic cytokine TGFß reduced adropin/ENHO expression in a JNK-dependent manner. Restoration of adropin signaling by therapeutic application of bioactive adropin34-76 peptides in turn inhibited TGFß-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices. Knockdown of GPR19, an adropin receptor, abrogated the antifibrotic effects of adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of adropin34-76 were functionally linked to deactivation of GLI1-dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo, and ex vivo using cultured human dermal fibroblasts, a sclGvHD mouse model, and precision-cut human skin slices. ChIP-seq confirmed adropin34-76-induced changes in TGFß/GLI1 signaling. Our study characterizes the TGFß-induced down-regulation of adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling.

Expression of NMU and NMUR1 in tryptase-positive mast cells and PBLs in allergic rhinitis patients' nasal mucosa.

BACKGROUND: The neuropeptide U (NMU) has been proven to elicit the release of mediators from mast cells (MCs) through its receptor NMUR1 in allergic inflammatory models. However, little is known about the correlations between NMU and MCs in human allergic rhinitis (AR). OBJECTIVE: The objective of this study is to investigate the expressions of NMU and NMUR1 in the tryptase + MCs and the peripheral blood leukocytes (PBLs) in human nasal mucosa with AR. METHODS: Specimens of nasal mucosa from patients with AR (n = 10) and control patients without AR (n = 8) were collected and soaked in frozen tissue liquid solution (OCT) in tum. Cryostat sections were prepared for immunofluorescence staining. Tryptase was used as a marker to detect mast cells and other tryptase + immune cells. The expression of NMU and NMUR1 was respectively determined by double staining using a confocal microscope. RESULTS: Neither NMU nor NMUR1 were detected in the tryptase + mast cells in the human nasal mucosa. To our surprise, both NMU and NMUR1 were co-expressed with tryptase in the PBLs within peripheral blood vessels in AR and controls. CONCLUSION: Our findings showed that NMU could not influence human nasal tryptase + mast cells directly through NMUR1 in AR. The co-expression of both NMU and NMUR1 with tryptase in the PBLs provided new insight into the potential roles of NMU and tryptase in the circulation PBLs, and the infiltrated PBLs may promote nasal allergic inflammation by producing tryptase and NMU.

Neuromedin U regulates the anti-tumor activity of CD8+ T cells and glycolysis of tumor cells in the tumor microenvironment of pancreatic ductal adenocarcinoma in an NMUR1-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis, which is lethal in approximately 90% of cases despite advanced standard therapies. A typical feature of PDAC is the immunosuppressive tumor microenvironment with multiple immunosuppressive factors including neurotransmitters. Recently, neuromedin U (NMU), a highly conserved neuropeptide with many physiological functions, has attracted attention for its roles in tumorigenesis and metastasis in several types of cancers. However, whether NMU affects PDAC progression remains unclear. In this study, using an orthotopic mouse model of PDAC in combination with bioinformatics analysis, we found that NMU was upregulated in tumor tissues from the patients with PDAC and positively correlated with a poor prognosis of the disease. Interestingly, knockout of the Nmu gene in mice enhanced the anti-tumor functions of tumor-infiltrating CD8+ T cells in an NMU receptor 1-dependent manner. Additionally, NMU promoted the glycolytic metabolism of mouse PDAC tumors. The activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH), pivotal enzymes involved in the regulation of lactate production, were markedly reduced in tumor tissues from NMU-knockout mice. In vitro the presence of LDHA inhibitor can reduce the production of lactic acid stimulated by NMU, which can increase the anti-tumor activity of CD8+ T cells. Moreover, treatment of the pancreatic cancer cells with a phosphoinositide 3-kinase (PI3K) inhibitor diminished NMU-induced lactate production and the activities of PK and LDH, suggesting that NMU might regulate glycolysis via the PI3K/AKT pathway.

Neurotransmitter Receptor HTR2B Regulates Lipid Metabolism to Inhibit Ferroptosis in Gastric Cancer.

Nerves can support tumor development by secreting neurotransmitters that promote cancer cell proliferation and invasion. 5-Hydroxytryptamine (5-HT) is a critical neurotransmitter in the gastrointestinal nervous system, and 5-HT signaling has been shown to play a role in tumorigenesis. Here, we found that expression of the 5-HT receptor HTR2B was significantly elevated in human gastric adenocarcinoma tissues compared with nontumor tissues, and high HTR2B expression corresponded to shorter patient survival. Both 5-HT and a specific HTR2B agonist enhanced gastric adenocarcinoma cell viability under metabolic stress, reduced cellular and lipid reactive oxygen species, and suppressed ferroptosis; conversely, HTR2B loss or inhibition with a selective HTR2B antagonist yielded the inverse tumor suppressive effects. In a patient-derived xenograft tumor model, HTR2B-positive tumors displayed accelerated growth, which was inhibited by HTR2B antagonists. Single-cell analysis of human gastric adenocarcinoma tissues revealed enrichment of PI3K/Akt/mTOR and fatty acid metabolism-related gene clusters in cells expressing HTR2B compared with HTR2B-negative cells. Mechanistically, HTR2B cooperated with Fyn to directly regulate p85 activity and trigger the PI3K/Akt/mTOR signaling pathway, which led to increased expression of HIF1α and ABCD1 along with decreased levels of lipid peroxidation and ferroptosis. Together, these findings demonstrate that HTR2B activity modulates PI3K/Akt/mTOR signaling to stimulate gastric cancer cell survival and indicate that HTR2B expression could be a potential prognostic biomarker in patients with gastric cancer. SIGNIFICANCE: Nerve cancer cross-talk mediated by HTR2B inhibits lipid peroxidation and ferroptosis in gastric cancer cells and promotes viability under metabolic stress, resulting in increased tumor growth and decreased patient survival.

Expression of G protein-coupled receptor GPR19 in normal and neoplastic human tissues.

Little is known about the expression of the orphan G protein-coupled receptor GPR19 at the protein level. Therefore, we developed a rabbit antibody, targeting human GPR19. After verification of the antibody specificity using GPR19-expressing cell lines and a GPR19-specific siRNA, the antibody was used for immunohistochemical staining of a variety of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. In normal tissues, GPR19 expression was detected in a distinct cell population within the cortex, in single cells of the pancreatic islets, in intestinal ganglia, gastric chief cells, and in endocrine cells of the bronchial tract, the gastrointestinal tract, and the prostate. Among the 30 different tumour entities investigated, strong GPR19 expression was found in adenocarcinomas, typical and atypical carcinoids of the lung, and small cell lung cancer. To a lesser extent, the receptor was also present in large cell neuroendocrine carcinomas of the lung, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, and a subpopulation of pancreatic neuroendocrine neoplasms. In lung tumours, a negative correlation with the expression of the proliferation marker Ki-67 and a positive interrelationship with patient survival was observed. Overall, our results indicate that in adenocarcinomas and neuroendocrine tumours of the lung GPR19 may serve as a suitable diagnostic or therapeutic target.

Human Dental Pulp Stem Cells Are Subjected to Metabolic Reprogramming and Repressed Proliferation and Migration by the Sympathetic Nervous System via α1B-Adrenergic Receptor.

INTRODUCTION: Human dental pulp stem cells (hDPSCs) reside in specialized microenvironments in the dental pulp, termed "niches," which are composed of diverse cellular components including nerves. Sensory nerves can positively regulate the expansion and differentiation of pulp cells, while the biological effects of the sympathetic nervous system (SNS) on hDPSCs remain elusive. This study is devoted to investigating the effects and underlying mechanisms of the SNS on the proliferation and migration of hDPSCs. METHODS: The distribution of sympathetic nerve fibers in human dental pulp was examined by immunofluorescence staining of tyrosine hydroxylase. The concentration of norepinephrine in healthy and carious human dental pulp tissues was detected using enzyme-linked immunosorbent assay. RNA-sequencing was applied to identify the dominant sympathetic neurotransmitter receptor in hDPSCs. Seahorse metabolic assay, adenosine triphosphate assay, lactate assay, and mitochondrial DNA copy number were performed to determine the level of glycometabolism. Transwell assay, wound healing assay, 5-ethynyl-2'-deoxyuridine staining assay, cell cycle assay, and Cell Counting Kit-8 assay were conducted to analyze the migratory and proliferative capacities of hDPSCs. RESULTS: Sprouting of sympathetic nerve fibers and an increased concentration of norepinephrine were observed in inflammatory pulp tissues. Sympathetic nerve fibers were mainly distributed along blood vessels, and aldehyde dehydrogenase 1-positive hDPSCs resided in close proximity to neurovascular bundles. ADRA1B was identified as the major sympathetic neurotransmitter receptor expressed in hDPSCs, and its expression was enhanced in inflammatory pulp tissues. In addition, the SNS inhibited the proliferation and migration of hDPSCs through metabolic reprogramming via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways. CONCLUSIONS: This study demonstrates that the SNS can shift the metabolism of hDPSCs from oxidative phosphorylation to anaerobic glycolysis via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways, thereby inhibiting the proliferative and migratory abilities of hDPSCs. This metabolic shift may facilitate the maintenance of the quiescent state of hDPSCs.

A conditional strategy for cell-type-specific labeling of endogenous excitatory synapses in Drosophila.

Chemical neurotransmission occurs at specialized contacts where neurotransmitter release machinery apposes neurotransmitter receptors to underlie circuit function. A series of complex events underlies pre- and postsynaptic protein recruitment to neuronal connections. To better study synaptic development in individual neurons, we need cell-type-specific strategies to visualize endogenous synaptic proteins. Although presynaptic strategies exist, postsynaptic proteins remain less studied because of a paucity of cell-type-specific reagents. To study excitatory postsynapses with cell-type specificity, we engineered dlg1[4K], a conditionally labeled marker of Drosophila excitatory postsynaptic densities. With binary expression systems, dlg1[4K] labels central and peripheral postsynapses in larvae and adults. Using dlg1[4K], we find that distinct rules govern postsynaptic organization in adult neurons, multiple binary expression systems can concurrently label pre- and postsynapse in a cell-type-specific manner, and neuronal DLG1 can sometimes localize presynaptically. These results validate our strategy for conditional postsynaptic labeling and demonstrate principles of synaptic organization.

Gradients of neurotransmitter receptor expression in the macaque cortex.

Dynamics and functions of neural circuits depend on interactions mediated by receptors. Therefore, a comprehensive map of receptor organization across cortical regions is needed. In this study, we used in vitro receptor autoradiography to measure the density of 14 neurotransmitter receptor types in 109 areas of macaque cortex. We integrated the receptor data with anatomical, genetic and functional connectivity data into a common cortical space. We uncovered a principal gradient of receptor expression per neuron. This aligns with the cortical hierarchy from sensory cortex to higher cognitive areas. A second gradient, driven by serotonin 5-HT1A receptors, peaks in the anterior cingulate, default mode and salience networks. We found a similar pattern of 5-HT1A expression in the human brain. Thus, the macaque may be a promising translational model of serotonergic processing and disorders. The receptor gradients may enable rapid, reliable information processing in sensory cortical areas and slow, flexible integration in higher cognitive areas.

GPR19 Coordinates Multiple Molecular Aspects of Stress Responses Associated with the Aging Process.

G protein-coupled receptors (GPCRs) play a significant role in controlling biological paradigms such as aging and aging-related disease. We have previously identified receptor signaling systems that are specifically associated with controlling molecular pathologies associated with the aging process. Here, we have identified a pseudo-orphan GPCR, G protein-coupled receptor 19 (GPR19), that is sensitive to many molecular aspects of the aging process. Through an in-depth molecular investigation process that involved proteomic, molecular biological, and advanced informatic experimentation, this study found that the functionality of GPR19 is specifically linked to sensory, protective, and remedial signaling systems associated with aging-related pathology. This study suggests that the activity of this receptor may play a role in mitigating the effects of aging-related pathology by promoting protective and remedial signaling systems. GPR19 expression variation demonstrates variability in the molecular activity in this larger process. At low expression levels in HEK293 cells, GPR19 expression regulates signaling paradigms linked with stress responses and metabolic responses to these. At higher expression levels, GPR19 expression co-regulates systems involved in sensing and repairing DNA damage, while at the highest levels of GPR19 expression, a functional link to processes of cellular senescence is seen. In this manner, GPR19 may function as a coordinator of aging-associated metabolic dysfunction, stress response, DNA integrity management, and eventual senescence.

Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket.

Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain dicarboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al., 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both dicarboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore-gating modulation. KEY POINTS: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent in mediating compound-elicited modulation. We propose that the two sites in the extracellular domain are involved 'in series', a mechanism which may have relevance for eukaryote receptors. We show that short-chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand-gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures. We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH. We report a caesium to sodium permeability ratio (PCs /PNa ) of 0.54 for GLIC ion pore.

Relationships between neurotransmitter receptor densities and expression levels of their corresponding genes in the human hippocampus.

Neurotransmitter receptors are key molecules in signal transmission, their alterations are associated with brain dysfunction. Relationships between receptors and their corresponding genes are poorly understood, especially in humans. We combined in vitro receptor autoradiography and RNA sequencing to quantify, in the same tissue samples (7 subjects), the densities of 14 receptors and expression levels of their corresponding 43 genes in the Cornu Ammonis (CA) and dentate gyrus (DG) of human hippocampus. Significant differences in receptor densities between both structures were found only for metabotropic receptors, whereas significant differences in RNA expression levels mostly pertained ionotropic receptors. Receptor fingerprints of CA and DG differ in shapes but have similar sizes; the opposite holds true for their "RNA fingerprints", which represent the expression levels of multiple genes in a single area. In addition, the correlation coefficients between receptor densities and corresponding gene expression levels vary widely and the mean correlation strength was weak-to-moderate. Our results suggest that receptor densities are not only controlled by corresponding RNA expression levels, but also by multiple regionally specific post-translational factors.

Ketamine preservative benzethonium chloride potentiates hippocampal synaptic transmission and binds neurotransmitter receptors and transporters.

Benzethonium chloride (BZT) is an excipient used in numerous products including (R,S)-ketamine (ketamine) drug formulations for human and veterinary use. Emerging evidence indicates BZT is pharmacologically active. BZT may therefore contribute to some of the clinical or preclinical effects observed with ketamine. In the present study, we evaluated: (i) the affinity of BZT for neurotransmitter receptors and transporters, (ii) the effects of BZT on hippocampal synaptic transmission in vitro, and (iii) plasma and brain concentrations of BZT following its intraperitoneal administration to male CD1 mice. Radioligand binding assays determined the affinity of BZT for neurotransmitter targets. Effects of BZT on field excitatory postsynaptic potentials (fEPSPs) were established via electrophysiological recordings from slices collected from male C57BL/6J mice. The binding assays revealed that BZT binds to numerous receptors (e.g., σ2 Ki = 7 nM) and transporters (e.g., dopamine transporter Ki = 545 nM). Bath application of BZT potentiated hippocampal fEPSPs in mouse hippocampal slices with an EC50 of 2.03 nM. Following intraperitoneal administration, BZT was detected in the plasma, but not in the brain of mice. These data highlight that studies measuring peripheral endpoints or directly exposing systems, in vitro, intracerebroventricularly, or intracortically, to BZT-containing formulations should account for the direct effects of BZT. Our findings also suggest that earlier data attributing pharmacological effects to ketamine may be confounded by BZT and that additional investigation into the functional impact of BZT is warranted. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

Ligand recognition and activation of neuromedin U receptor 2.

Neuromedin U receptor 2 (NMU2), an emerging attractive target for treating obesity, has shown the capability in reducing food intake and regulating energy metabolism when activated. However, drug development of NMU2 was deferred partially due to the lack of structural information. Here, we present the cryo-electron microscopy (cryo-EM) structure of NMU2 bound to the endogenous agonist NmU-25 and Gi1 at 3.3 Å resolution. Combined with functional and computational data, the structure reveals the key factors that govern the recognition and selectivity of peptide agonist as well as non-peptide antagonist, providing the structural basis for design of novel and highly selective drugs targeting NMU2. In addition, a 25-degree rotation of Gi protein in reference to NMU2 is also observed compared in other structures of class A GPCR-Gi complexes, suggesting heterogeneity in the processes of G protein-coupled receptors (GPCRs) activation and G protein coupling.

Intersection of the Orphan G Protein-Coupled Receptor, GPR19, with the Aging Process.

G protein-coupled receptors (GPCRs) represent one of the most functionally diverse classes of transmembrane proteins. GPCRs and their associated signaling systems have been linked to nearly every physiological process. They also constitute nearly 40% of the current pharmacopeia as direct targets of remedial therapies. Hence, their place as a functional nexus in the interface between physiological and pathophysiological processes suggests that GPCRs may play a central role in the generation of nearly all types of human disease. Perhaps one mechanism through which GPCRs can mediate this pivotal function is through the control of the molecular aging process. It is now appreciated that, indeed, many human disorders/diseases are induced by GPCR signaling processes linked to pathological aging. Here we discuss one such novel member of the GPCR family, GPR19, that may represent an important new target for novel remedial strategies for the aging process. The molecular signaling pathways (metabolic control, circadian rhythm regulation and stress responsiveness) associated with this recently characterized receptor suggest an important role in aging-related disease etiology.

Neuromedin U-deficient rats do not lose body weight or food intake.

Studies in genetically modified mice establish that essential roles of endogenous neuromedin U (NMU) are anorexigenic function and metabolic regulation, indicating that NMU is expected to be a potential target for anti-obesity agents. However, in central administration experiments in rats, inconsistent results have been obtained, and the essential role of NMU energy metabolism in rats remain unclear. This study aims to elucidate the role of endogenous NMU in rats. We generated NMU knockout (KO) rats that unexpectedly showed no difference in body weight, adiposity, circulating metabolic markers, body temperature, locomotor activity, and food consumption in both normal and high fat chow feeding. Furthermore, unlike reported in mice, expressions of Nmu and NMU receptor type 2 (Nmur2) mRNA were hardly detectable in the rat hypothalamic nuclei regulating feeding and energy metabolism, including the arcuate nucleus and paraventricular nucleus, while Nmu was expressed in pars tuberalis and Nmur2 was expressed in the ependymal cell layer of the third ventricle. These results indicate that the species-specific expression pattern of Nmu and Nmur2 may allow NMU to have distinct functions across species, and that endogenous NMU does not function as an anorexigenic hormone in rats.

Correspondence between gene expression and neurotransmitter receptor and transporter density in the human brain.

Neurotransmitter receptors modulate signaling between neurons. Thus, neurotransmitter receptors and transporters play a key role in shaping brain function. Due to the lack of comprehensive neurotransmitter receptor/transporter density datasets, microarray gene expression measuring mRNA transcripts is often used as a proxy for receptor densities. In the present report, we comprehensively test the spatial correlation between gene expression and protein density for a total of 27 neurotransmitter receptors, receptor binding-sites, and transporters across 9 different neurotransmitter systems, using both PET and autoradiography radioligand-based imaging modalities. We find poor spatial correspondences between gene expression and density for all neurotransmitter receptors and transporters except four single-protein metabotropic receptors (5-HT1A, CB1, D2, and MOR). These expression-density associations are related to gene differential stability and can vary between cortical and subcortical structures. Altogether, we recommend using direct measures of receptor and transporter density when relating neurotransmitter systems to brain structure and function.

Molecular characterization and functional roles for Vibrio alginolyticus resistance of an octopamine/tyramine receptor of the white shrimp, Litopenaeus vannamei.

Octopamine and Tyramine are biogenic amines that have been demonstrated to play an important immunological role in white shrimp, Litopenaeus vannamei. G protein-coupled receptors, known as seven-transmembrane domain receptors, are a variety of neurotransmitter receptors which are sensitive to biogenic amines for initiating the cell signaling pathway. In present study, we cloned and characterized an octopamine/tyramine receptor (LvOA/TA-R) from the hemocytes of L. vannamei, with a 1194 b.p. open reading frame that encodes 398 amino acids. Several bioinformatics analyses indicated that LvOA/TA-R had seven conserved hydrophobic transmembrane domains. The phylogenetic analysis and multiple sequence alignment indicated that LvOA/TA-R was orthologous to the OA/TA receptor of tiger shrimp, P. monodon. LvOA/TA-R was expressed in hemocytes and nervous tissue including circumoesphageal connective tissue and the thoracic and abdominal ganglia. Significant increases in LvOA/TA-R occurred in hemocytes of L. vannamei under Vibrio alginolyticus infection within 30-60 min of infection. Here, we demonstrated that LvOA/TA-R expression is upregulated in response to Vibrio alginolyticus infection and appears to be functionally responsible for the observed immune response. These results suggest that LvOA/TA-R mediates regulation of immunity, which promotes the resistance of L. vannamei to V. alginolyticus.

Vesicular Glutamate Release from Feeder-FreehiPSC-Derived Neurons.

Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons.

Neuroprotective Effects of σ2R/TMEM97 Receptor Modulators in the Neuronal Model of Huntington's Disease.

Huntington's disease (HD) is a genetic neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene that encodes for an expanded polyglutamine (polyQ) repeat in exon-1 of the human mutant huntingtin (mHTT) protein. The presence of this polyQ repeat results in neuronal degeneration, for which there is no cure or treatment that modifies disease progression. In previous studies, we have shown that small molecules that bind selectively to σ2R/TMEM97 can have significant neuroprotective effects in models of Alzheimer's disease, traumatic brain injury, and several other neurodegenerative diseases. In the present work, we extend these investigations and show that certain σ2R/TMEM97-selective ligands decrease mHTT-induced neuronal toxicity. We first synthesized a set of compounds designed to bind to σ2R/TMEM97 and determined their binding profiles (Ki values) for σ2R/TMEM97 and other proteins in the central nervous system. Modulators with high affinity and selectivity for σ2R/TMEM97 were then tested in our HD cell model. Primary cortical neurons were cultured in vitro for 7 days and then co-transfected with either a normal HTT construct (Htt N-586-22Q/GFP) or the mHTT construct Htt-N586-82Q/GFP. Transfected neurons were treated with either σ2R/TMEM97 or σ1R modulators for 48 h. After treatment, neurons were fixed and stained with Hoechst, and condensed nuclei were quantified to assess cell death in the transfected neurons. Significantly, σ2R/TMEM97 modulators reduce the neuronal toxicity induced by mHTT, and their neuroprotective effects are not blocked by NE-100, a selective σ1R antagonist known to block neuroprotection by σ1R ligands. These results indicate for the first time that σ2R/TMEM97 modulators can protect neurons from mHTT-induced neuronal toxicity, suggesting that targeting σ2R/TMEM97 may lead to a novel therapeutic approach to treat patients with HD.

Local accumulation times in a diffusion-trapping model of receptor dynamics at proximal axodendritic synapses.

The lateral diffusion and trapping of neurotransmitter receptors within the postsynaptic membrane of a neuron play a key role in determining synaptic strength and plasticity. Trapping is mediated by the reversible binding of receptors to scaffolding proteins (slots) within a synapse. In this paper we introduce a method for analyzing the transient dynamics of proximal axodendritic synapses in a diffusion-trapping model of receptor trafficking. Given a population of spatially distributed synapses, each of which has a fixed number of slots, we calculate the rate of relaxation to the steady-state distribution of bound slots (synaptic weights) in terms of a set of local accumulation times. Assuming that the rates of exocytosis and endocytosis are sufficiently slow, we show that the steady-state synaptic weights are independent of each other (purely local). On the other hand, the local accumulation time of a given synapse depends on the number of slots and the spatial location of all the synapses, indicating a form of transient heterosynaptic plasticity. This suggests that local accumulation time measurements could provide useful information regarding the distribution of synaptic weights within a dendrite.