Serum autoantibodies against α7-nicotinic receptors in subgroups of patients with bipolar disorder or schizophrenia: clinical features and link with peripheral inflammation.

There is growing evidence that autoantibodies (AAbs) against proteins expressed in the brain are playing an important role in neurological and psychiatric disorders. Here, we explore the presence and the role of peripheral AAbs to the α7-nicotinic acetylcholine receptor (nAChR) in inflammatory subgroups of psychiatric patients with bipolar disorder (BD) or schizophrenia (SCZ) and healthy controls. We have identified a continuum of AAb levels in serum when employing a novel ELISA technique, with a significant elevation in patients compared to controls. Using unsupervised two-step clustering to stratify all the subjects according to their immuno-inflammatory background, we delineate one subgroup consisting solely of psychiatric patients with severe symptoms, high inflammatory profile, and significantly increased levels of anti-nAChR AAbs. In this context, we have used monoclonal mouse anti-human α7-nAChR antibodies (α7-nAChR-mAbs) and shown that TNF-α release was enhanced upon LPS stimulation in macrophages pre-incubated with α7-nAChR-mAbs compared to the use of an isotype control. These findings provide a basis for further study of circulating nicotinic AAbs, and the inflammatory profile observed in patients with major mood and psychotic disorders.

Functional maturation of human iPSC-derived pyramidal neurons in vivo is dependent on proximity with the host tissue.

Human induced pluripotent stem cells (hiPSCs) have been used extensively in vitro to model early events in neurodevelopment. Because of a number of shortcomings, previous work has established a potential to use these cells in vivo after transplantation into the mouse brain. Here, we describe a systematic approach for the analysis of transplanted hiPSC-derived neurons and glial cells over time in the mouse brain. Using functional two-photon imaging of GCaMP6f- expressing human neural cells, we define and quantify the embryonic-like features of their spontaneous activity. This is substantiated by detailed electron microscopy (EM) of the graft. We relate this to the synaptic development the neurons undergo up to 7 months in vivo. This system can now be used further for the genetic or experimental manipulation of developing hiPSC-derived cells addressing neurodevelopmental diseases like schizophrenia or Autism Spectrum Disorder.

An original potentiating mechanism revealed by the cryo-EM structures of the human α7 nicotinic receptor in complex with nanobodies.

The human α7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest in designing ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of α7 ligands, we recently generated two nanobodies named E3 and C4, acting as positive allosteric modulator and silent allosteric ligand, respectively. Here, we solved the cryo-electron microscopy structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an agonist-bound conformation of the extracellular domain in the absence of an orthosteric agonist, and mutational analysis shows a key contribution of an N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation that opens new avenues for drug design.

Prolonged nicotine exposure reduces aversion to the drug in mice by altering nicotinic transmission in the interpeduncular nucleus.

Nicotine intake is likely to result from a balance between the rewarding and aversive properties of the drug, yet the individual differences in neural activity that control aversion to nicotine and their adaptation during the addiction process remain largely unknown. Using a two-bottle choice experiment, we observed considerable heterogeneity in nicotine-drinking profiles in isogenic adult male mice, with about half of the mice persisting in nicotine consumption even at high concentrations, whereas the other half stopped consuming. We found that nicotine intake was negatively correlated with nicotine-evoked currents in the interpeduncular nucleus (IPN), and that prolonged exposure to nicotine, by weakening this response, decreased aversion to the drug, and hence boosted consumption. Lastly, using knock-out mice and local gene re-expression, we identified ß4-containing nicotinic acetylcholine receptors of IPN neurons as molecular and cellular correlates of nicotine aversion. Collectively, our results identify the IPN as a substrate for individual variabilities and adaptations in nicotine consumption.

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human α7-nAChR were generated through alpaca immunization with cells expressing a human α7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4ß2 and α3ß4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

Developmental Changes of Human Neural Progenitor Cells Grafted into the Ventricular System and Prefrontal Cortex of Mouse Brain in Utero.

The transplantation of neural progenitors into a host brain represents a useful tool to evaluate the involvement of cell-autonomous processes and host local cues in the regulation of neuronal differentiation during the development of the mammalian brain. Human brain development starts at the embryonic stages, in utero, with unique properties at its neotenic stages. We analyzed the engraftment and differentiation of human neuronal progenitor cells (hNPCs) transplanted in utero into the mouse brain. The influence of the environment was studied by transplanting human NPCs within the lateral ventricles (LV), compared with the prefrontal cortex (PFC) of immunocompetent mice. We developed a semi-automated method to accurately quantify the number of cell bodies and the distribution of neuronal projections among the different mouse brain structures, at 1 and 3 months post-transplantation (MPT). Our data show that human NPCs can differentiate between immature "juvenile" neurons and more mature pyramidal cells in a reproducible manner. Depending on the injection site, LV vs. PFC, specific fetal local environments could modify the synaptogenesis processes while maintaining human neoteny. The use of immunocompetent mice as host species allows us to investigate further neuropathological conditions making use of all of the engineered mouse models already available.

Evidence for a protective effect of the loss of α4-containing nicotinic acetylcholine receptors on Aß-related neuropathology in Tg2576 mice.

Introduction: Loss of cholinergic neurons as well as α4ß2* (* = containing) nicotinic acetylcholine receptors (nAChRs) is a prominent feature of Alzheimer's disease (AD). Specifically, amyloid ß (Aß), the principal pathogenic factor of AD, is a high affinity ligand for nAChRs. Yet, the pathophysiological role of nAChRs in AD is not well established. Methods: In the present study, we have investigated the effects of the loss of α4* nAChRs on the histological alterations of the Tg2576 mouse model of AD (APPswe) crossing hemizygous APPswe mice with mice carrying the genetic inactivation of α4 nAChR subunit (α4KO). Results: A global decrease in Aß plaque load was observed in the forebrain of APPswe/α4KO mice in comparison with APPswe mice, that was particularly marked in neocortex of 15 month-old mice. At the same age, several alterations in synaptophysin immunoreactivity were observed in cortico-hippocampal regions of APPswe mice that were partially counteracted by α4KO. The analysis of the immunoreactivity of specific astroglia (glial fibrillary acidic protein, GFAP) and microglia (ionized calcium-binding adapter molecule, Iba1) markers showed an increase in the number as well as in the area occupied by these cells in APPswe mice that were partially counteracted by α4KO. Conclusion: Overall, the present histological study points to a detrimental role of α4* nAChRs that may be specific for Aß-related neuropathology.

Impact of the gut microbiome on nicotine's motivational effects and glial cells in the ventral tegmental area in male mice.

A link between gut dysbiosis and the pathogenesis of brain disorders has been identified. A role for gut bacteria in drug reward and addiction has been suggested but very few studies have investigated their impact on brain and behavioral responses to addictive drugs so far. In particular, their influence on nicotine's addiction-like processes remains unknown. In addition, evidence shows that glial cells shape the neuronal activity of the mesolimbic system but their regulation, within this system, by the gut microbiome is not established. We demonstrate that a lack of gut microbiota in male mice potentiates the nicotine-induced activation of sub-regions of the mesolimbic system. We further show that gut microbiota depletion enhances the response to nicotine of dopaminergic neurons of the posterior ventral tegmental area (pVTA), and alters nicotine's rewarding and aversive effects in an intra-VTA self-administration procedure. These effects were not associated with gross behavioral alterations and the nicotine withdrawal syndrome was not impacted. We further show that depletion of the gut microbiome modulates the glial cells of the mesolimbic system. Notably, it increases the number of astrocytes selectively in the pVTA, and the expression of postsynaptic density protein 95 in both VTA sub-regions, without altering the density of the astrocytic glutamatergic transporter GLT1. Finally, we identify several sub-populations of microglia in the VTA that differ between its anterior and posterior sub-parts, and show that they are re-organized in conditions of gut microbiota depletion. The present study paves the way for refining our understanding of the pathophysiology of nicotine addiction.

The Nicotinic Receptor Polymorphism rs16969968 Is Associated with Airway Remodeling and Inflammatory Dysregulation in COPD Patients.

Genome-wide association studies unveiled the associations between the single nucleotide polymorphism rs16969968 of CHRNA5, encoding the nicotinic acetylcholine receptor alpha5 subunit (α5SNP), and nicotine addiction, cancer, and COPD independently. Here, we investigated α5SNP-induced epithelial remodeling and inflammatory response in human COPD airways. We included 26 α5SNP COPD patients and 18 wild-type α5 COPD patients in a multi-modal study. A comparative histologic analysis was performed on formalin-fixed paraffin-embedded lung tissues. Isolated airway epithelial cells from bronchial brushings were cultivated in the air-liquid interface. Broncho-alveolar fluids were collected to detect inflammatory mediators. Ciliogenesis was altered in α5SNP COPD bronchial and bronchiolar epithelia. Goblet cell hyperplasia was exacerbated in α5SNP small airways. The broncho-alveolar fluids of α5SNP COPD patients exhibited an increase in inflammatory mediators. The involvement of the rs16969968 polymorphism in airway epithelial remodeling and related inflammatory response in COPD prompts the development of innovative personalized diagnostic and therapeutic strategies.

Divergent Roles of α5 and ß4 Nicotinic Receptor Subunits in Food Reward and Nicotine-induced Weight Loss in Male Mice.

A major obstacle to successful smoking cessation is the prospect of weight gain. Despite a clear relationship between cigarette smoking and body weight, surprisingly little is known about the physiological and molecular mechanism by which nicotine affects energy homeostasis and food-motivated behaviors. Here we use loss-of-function mouse models to demonstrate that 2 nicotinic acetylcholine receptor (nAChR) subunits encoded by the CHRNA5-CHRNA3-CHRNB4 gene cluster, α5 and ß4, exhibit divergent roles in food reward. We also reveal that ß4-containing nAChRs are essential for the weight-lowering effects of nicotine in diet-induced obese mice. Finally, our data support the notion of crosstalk between incretin biology and nAChR signaling, as we demonstrate that the glycemic benefits of glucagon-like peptide-1 receptor activation partially relies on ß4-containing nAChRs. Together, these data encourage further research into the role of cholinergic neurotransmission in regulating food reward and the translational pursuit of site-directed targeting of ß4-containing nAChRs for treatment of metabolic disease.

Nicotinic receptors as SARS-CoV-2 spike co-receptors?

Nicotinic acetylcholine receptors (nAChRs) play an important role in homeostasis and respiratory diseases. Controversies regarding the association between COVID-19 hospitalizations and smoking suggest that nAChRs may contribute to SARS-CoV-2 respiratory syndrome. We recently detailed the expression and localization of all nAChR subunits in the human lung. Since virus association with nAChRs has been shown in the past, we hypothesize that nAChR subunits act as SARS-CoV-2 Spike co-receptors. Based on sequence alignment analysis, we report domains of high molecular similarities in nAChRs with the binding domain of hACE2 for SARS-CoV-2 Spike protein. This hypothesis supported by in silico pilot data provides a rational for the modelling and the in vitro experimental validation of the interaction between SARS-CoV-2 and the nAChRs.

An innate contribution of human nicotinic receptor polymorphisms to COPD-like lesions.

Chronic Obstructive Pulmonary Disease is a generally smoking-linked major cause of morbidity and mortality. Genome-wide Association Studies identified a locus including a non-synonymous single nucleotide polymorphism in CHRNA5, rs16969968, encoding the nicotinic acetylcholine receptor α5 subunit, predisposing to both smoking and Chronic Obstructive Pulmonary Disease. Here we report that nasal polyps from rs16969968 non-smoking carriers exhibit airway epithelium remodeling and inflammation. These hallmarks of Chronic Obstructive Pulmonary Disease occur spontaneously in mice expressing human rs16969968. They are significantly amplified after exposure to porcine pancreatic elastase, an emphysema model, and to oxidative stress with a polymorphism-dependent alteration of lung function. Targeted rs16969968 expression in epithelial cells leads to airway remodeling in vivo, increased proliferation and production of pro-inflammatory cytokines through decreased calcium entry and increased adenylyl-cyclase activity. We show that rs16969968 directly contributes to Chronic Obstructive Pulmonary Disease-like lesions, sensitizing the lung to the action of oxidative stress and injury, and represents a therapeutic target.

Cholinergic modulation of hierarchical inhibitory control over cortical resting state dynamics: Local circuit modeling of schizophrenia-related hypofrontality.

Nicotinic acetylcholine receptors (nAChRs) modulate the cholinergic drive to a hierarchy of inhibitory neurons in the superficial layers of the PFC, critical to cognitive processes. It has been shown that genetic deletions of the various types of nAChRs impact the properties of ultra-slow transitions between high and low PFC activity states in mice during quiet wakefulness. The impact characteristics depend on specific interneuron populations expressing the manipulated receptor subtype. In addition, recent data indicate that a genetic mutation of the α5 nAChR subunit, located on vasoactive intestinal polypeptide (VIP) inhibitory neurons, the rs16969968 single nucleotide polymorphism (α5 SNP), plays a key role in the hypofrontality observed in schizophrenia patients carrying the SNP. Data also indicate that chronic nicotine application to α5 SNP mice relieves the hypofrontality. We developed a computational model to show that the activity patterns recorded in the genetically modified mice can be explained by changes in the dynamics of the local PFC circuit. Notably, our model shows that these altered PFC circuit dynamics are due to changes in the stability structure of the activity states. We identify how this stability structure is differentially modulated by cholinergic inputs to the parvalbumin (PV), somatostatin (SOM) or the VIP inhibitory populations. Our model uncovers that a change in amplitude, but not duration of the high activity states can account for the lowered pyramidal (PYR) population firing rates recorded in α5 SNP mice. We demonstrate how nicotine-induced desensitization and upregulation of the ß2 nAChRs located on SOM interneurons, as opposed to the activation of α5 nAChRs located on VIP interneurons, is sufficient to explain the nicotine-induced activity normalization in α5 SNP mice. The model further implies that subsequent nicotine withdrawal may exacerbate the hypofrontality over and beyond one caused by the SNP mutation.

Inhibitory control of synaptic signals preceding locomotion in mouse frontal cortex.

The frontal cortex is essential for organizing voluntary movement. The secondary motor cortex (MOs) is a frontal subregion thought to integrate internal and external inputs before motor action. However, how excitatory and inhibitory synaptic inputs to MOs neurons are integrated preceding movement remains unclear. Here, we address this question by performing in vivo whole-cell recordings from MOs neurons of head-fixed mice moving on a treadmill. We find that principal neurons produce slowly increasing membrane potential and spike ramps preceding spontaneous running. After goal-directed training, ramps show larger amplitudes and accelerated kinetics. Chemogenetic suppression of interneurons combined with modeling suggests that the interplay between parvalbumin-positive (PV+) and somatostatin-positive (SOM+) interneurons, along with principal neuron recurrent connectivity, shape ramping signals. Plasticity of excitatory synapses on SOM+ interneurons can explain the ramp acceleration after training. Altogether, our data reveal that local interneurons differentially control task-dependent ramping signals when MOs neurons integrate inputs preceding movement.

CD147 Promotes Tumor Lymphangiogenesis in Melanoma via PROX-1.

Malignant melanoma is one of the most aggressive skin cancers and is characterized by early lymph node metastasis and the capacity to develop resistance to therapies. Hence, understanding the regulation of lymphangiogenesis through mechanisms contributing to lymphatic vessel formation represents a treatment strategy for metastatic cancer. We have previously shown that CD147, a transmembrane glycoprotein overexpressed in melanoma, regulates the angiogenic process in endothelial cells. In this study, we show a correlation between high CD147 expression levels and the number of lymphatic vessels expressing LYVE-1, Podoplanin, and VEGFR-3 in human melanoma lymph nodes. CD147 upregulates in vitro lymphangiogenesis and its related mediators through the PROX-1 transcription factor. In vivo studies in a melanoma model confirmed that CD147 is involved in metastasis through a similar mechanism as in vitro. This study, demonstrating the paracrine role of CD147 in the lymphangiogenesis process, suggests that CD147 could be a promising target for the inhibition of melanoma-associated lymphangiogenesis.

Chr15q25 Genetic Variant rs16969968 Alters Cell Differentiation in Respiratory Epithelia.

The gene cluster region, CHRNA3/CHRNA5/CHRNB4, encoding for nicotinic acetylcholine receptor (nAChR) subunits, contains several genetic variants linked to nicotine addiction and brain disorders. The CHRNA5 single-nucleotide polymorphism (SNP) rs16969968 is strongly associated with nicotine dependence and lung diseases. Using immunostaining studies on tissue sections and air-liquid interface airway epithelial cell cultures, in situ hybridisation, transcriptomic and cytokines detection, we analysed rs16969968 contribution to respiratory airway epithelial remodelling and modulation of inflammation. We provide cellular and molecular analyses which support the genetic association of this polymorphism with impaired ciliogenesis and the altered production of inflammatory mediators. This suggests its role in lung disease development.

Nicotine inhibits the VTA-to-amygdala dopamine pathway to promote anxiety.

Nicotine stimulates dopamine (DA) neurons of the ventral tegmental area (VTA) to establish and maintain reinforcement. Nicotine also induces anxiety through an as yet unknown circuitry. We found that nicotine injection drives opposite functional responses of two distinct populations of VTA DA neurons with anatomically segregated projections: it activates neurons that project to the nucleus accumbens (NAc), whereas it inhibits neurons that project to the amygdala nuclei (Amg). We further show that nicotine mediates anxiety-like behavior by acting on ß2-subunit-containing nicotinic acetylcholine receptors of the VTA. Finally, using optogenetics, we bidirectionally manipulate the VTA-NAc and VTA-Amg pathways to dissociate their contributions to anxiety-like behavior. We show that inhibition of VTA-Amg DA neurons mediates anxiety-like behavior, while their activation prevents the anxiogenic effects of nicotine. These distinct subpopulations of VTA DA neurons with opposite responses to nicotine may differentially drive the anxiogenic and the reinforcing effects of nicotine.

Alterations in nicotinic receptor alpha5 subunit gene differentially impact early and later stages of cocaine addiction: a translational study in transgenic rats and patients.

Cocaine addiction is a chronic and relapsing disorder with an important genetic component. Human candidate gene association studies showed that the single nucleotide polymorphism (SNP) rs16969968 in the α5 subunit (α5SNP) of nicotinic acetylcholine receptors (nAChRs), previously associated with increased tobacco dependence, was linked to a lower prevalence of cocaine use disorder (CUD). Three additional SNPs in the α5 subunit, previously shown to modify α5 mRNA levels, were also associated with CUD, suggesting an important role of the subunit in this pathology. To investigate the link between this subunit and CUD, we submitted rats knockout for the α5 subunit gene (α5KO), or carrying the α5SNP, to cocaine self-administration (SA) and showed that the acquisition of cocaine-SA was impaired in α5SNP rats while α5KO rats exhibited enhanced cocaine-induced relapse associated with altered neuronal activity in the nucleus accumbens. In addition, we observed in a human cohort of patients with CUD that the α5SNP was associated with a slower transition from first cocaine use to CUD. We also identified a novel SNP in the ß4 nAChR subunit, part of the same gene cluster in the human genome and potentially altering CHRNA5 expression, associated with shorter time to relapse to cocaine use in patients. In conclusion, the α5SNP is protective against CUD by influencing early stages of cocaine exposure while CHRNA5 expression levels may represent a biomarker for the risk to relapse to cocaine use. Drugs modulating α5 containing nAChR activity may thus represent a novel therapeutic strategy against CUD.

Nicotinic Receptor Subunits Atlas in the Adult Human Lung.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels responsible for rapid neural and neuromuscular signal transmission. Although it is well documented that 16 subunits are encoded by the human genome, their presence in airway epithelial cells (AECs) remains poorly understood, and contribution to pathology is mainly discussed in the context of cancer. We analysed nAChR subunit expression in the human lungs of smokers and non-smokers using transcriptomic data for whole-lung tissues, isolated large AECs, and isolated small AECs. We identified differential expressions of nAChRs in terms of detection and repartition in the three modalities. Smoking-associated alterations were also unveiled. Then, we identified an nAChR transcriptomic print at the single-cell level. Finally, we reported the localizations of detectable nAChRs in bronchi and large bronchioles. Thus, we compiled the first complete atlas of pulmonary nAChR subunits to open new avenues to further unravel the involvement of these receptors in lung homeostasis and respiratory diseases.