Rapid synaptic and gamma rhythm signature of mouse critical period plasticity.

Early-life experience enduringly sculpts thalamocortical (TC) axons and sensory processing. Here, we identify the very first synaptic targets that initiate critical period plasticity, heralded by altered cortical oscillations. Monocular deprivation (MD) acutely induced a transient (<3 h) peak in EEG γ-power (~40 Hz) specifically within the visual cortex, but only when the critical period was open (juvenile mice or adults after dark-rearing, Lynx1-deletion, or diazepam-rescued GAD65-deficiency). Rapid TC input loss onto parvalbumin-expressing (PV) inhibitory interneurons (but not onto nearby pyramidal cells) was observed within hours of MD in a TC slice preserving the visual pathway - again once critical periods opened. Computational TC modeling of the emergent γ-rhythm in response to MD delineated a cortical interneuronal gamma (ING) rhythm in networks of PV-cells bearing gap junctions at the start of the critical period. The ING rhythm effectively dissociated thalamic input from cortical spiking, leading to rapid loss of previously strong TC-to-PV connections through standard spike-timing-dependent plasticity rules. As a consequence, previously silent TC-to-PV connections could strengthen on a slower timescale, capturing the gradually increasing γ-frequency and eventual fade-out over time. Thus, ING enables cortical dynamics to transition from being dominated by the strongest TC input to one that senses the statistics of population TC input after MD. Taken together, our findings reveal the initial synaptic events underlying critical period plasticity and suggest that the fleeting ING accompanying a brief sensory perturbation may serve as a robust readout of TC network state with which to probe developmental trajectories.

Lynx1 and the family of endogenous mammalian neurotoxin-like proteins and their roles in modulating nAChR function.

The promise of nicotinic receptors as a therapeutic target has yet to be fully realized, despite solid data supporting their involvement in neurological and neuropsychiatric diseases. The reasons for this are likely complex and manifold, having to do with the widespread action of the cholinergic system and the biophysical mechanism of action of nicotinic receptors leading to fast desensitization and down-regulation. Conventional drug development strategies tend to focus on receptor subtype-specific action of candidate therapeutics, although the broad agonist, nicotine, is being explored in the clinic. The potential negative effects of nicotine make the search for alternate strategies warranted. Prototoxins are a promising yet little-explored avenue of nicotinic receptor drug development. Nicotinic receptors in the brain belong to a complex of proteins, including those that bind to the extracellular face of the receptor, as well as chaperones that bind the intracellular domain, etc. Lynx prototoxins have allosteric modularity effects on receptor function and number and have been implicated in complex in vivo processes such as neuroplasticity, learning, and memory. Their mechanism of action and binding specificity on sets of nAChR subtypes present intriguing possibilities for more efficacious and nuanced therapeutic targeting than nicotinic receptor subtypes alone. An allosteric drug may restrict its actions to physiologically relevant time points, which tend to be correlated with salient events which would be encoded into long-term memory storage. Rather than blanketing the brain with a steady and prolonged elevation of agonist, an allosteric nAChR compound could avoid side effects and loss of efficacy over time. This review details the potential strengths and challenges of prototoxin proteins as therapeutic targets, and some of the utility of such therapeutics based on the emerging understanding of cholinergic signaling in a growing number of complex neural processes.

Deep learning of spontaneous arousal fluctuations detects early cholinergic defects across neurodevelopmental mouse models and patients.

Neurodevelopmental spectrum disorders like autism (ASD) are diagnosed, on average, beyond age 4 y, after multiple critical periods of brain development close and behavioral intervention becomes less effective. This raises the urgent need for quantitative, noninvasive, and translational biomarkers for their early detection and tracking. We found that both idiopathic (BTBR) and genetic (CDKL5- and MeCP2-deficient) mouse models of ASD display an early, impaired cholinergic neuromodulation as reflected in altered spontaneous pupil fluctuations. Abnormalities were already present before the onset of symptoms and were rescued by the selective expression of MeCP2 in cholinergic circuits. Hence, we trained a neural network (ConvNetACh) to recognize, with 97% accuracy, patterns of these arousal fluctuations in mice with enhanced cholinergic sensitivity (LYNX1-deficient). ConvNetACh then successfully detected impairments in all ASD mouse models tested except in MeCP2-rescued mice. By retraining only the last layers of ConvNetACh with heart rate variation data (a similar proxy of arousal) directly from Rett syndrome patients, we generated ConvNetPatients, a neural network capable of distinguishing them from typically developing subjects. Even with small cohorts of rare patients, our approach exhibited significant accuracy before (80% in the first and second year of life) and into regression (88% in stage III patients). Thus, transfer learning across species and modalities establishes spontaneous arousal fluctuations combined with deep learning as a robust noninvasive, quantitative, and sensitive translational biomarker for the rapid and early detection of neurodevelopmental disorders before major symptom onset.

Aß1-42 Accumulation Accompanies Changed Expression of Ly6/uPAR Proteins, Dysregulation of the Cholinergic System, and Degeneration of Astrocytes in the Cerebellum of Mouse Model of Early Alzheimer Disease.

Alzheimer disease (AD) is a widespread neurodegenerative disease characterized by the accumulation of oligomeric toxic forms of ß-amyloid (Aß1-42) and dysfunction of the cholinergic system in the different brain regions. However, the exact mechanisms of AD pathogenesis and the role of the nicotinic acetylcholine receptors (nAChRs) in the disease progression remain unclear. Here, we revealed a decreased expression of a number of the Ly6/uPAR proteins targeting nAChRs in the cerebellum of 2xTg-AD mice (model of early AD) in comparison with non-transgenic mice both at mRNA and protein levels. We showed that co-localization of one of them, - neuromodulator Lynx1, with α7-nAChR was diminished in the vicinity of cerebellar astrocytes of 2xTg-AD mice, while Aß1-42 co-localization with this receptor present was increased. Moreover, the expression of anti-inflammatory transcription factor KLF4 regulating transcription of the Ly6/uPAR genes was decreased in the cerebellum of 2xTg-AD mice, while expression of inflammatory cytokine TNF-α was increased. Based on these data together with observed astrocyte degeneration in the cerebellum of 2xTg-AD mice, we suggest the mechanism by which expression of the Ly6/uPAR proteins upon Aß pathology results in dysregulation of the cholinergic system and particularly of α7-nAChR function in the cerebellum. This leads to enhanced neuroinflammation and cerebellar astrocyte degeneration.

α7- and α9-Containing Nicotinic Acetylcholine Receptors in the Functioning of Immune System and in Pain.

Nicotinic acetylcholine receptors (nAChRs) present as many different subtypes in the nervous and immune systems, muscles and on the cells of other organs. In the immune system, inflammation is regulated via the vagus nerve through the activation of the non-neuronal α7 nAChR subtype, affecting the production of cytokines. The analgesic properties of α7 nAChR-selective compounds are mostly based on the activation of the cholinergic anti-inflammatory pathway. The molecular mechanism of neuropathic pain relief mediated by the inhibition of α9-containing nAChRs is not fully understood yet, but the role of immune factors in this process is becoming evident. To obtain appropriate drugs, a search of selective agonists, antagonists and modulators of α7- and α9-containing nAChRs is underway. The naturally occurring three-finger snake α-neurotoxins and mammalian Ly6/uPAR proteins, as well as neurotoxic peptides α-conotoxins, are not only sophisticated tools in research on nAChRs but are also considered as potential medicines. In particular, the inhibition of the α9-containing nAChRs by α-conotoxins may be a pathway to alleviate neuropathic pain. nAChRs are involved in the inflammation processes during AIDS and other viral infections; thus they can also be means used in drug design. In this review, we discuss the role of α7- and α9-containing nAChRs in the immune processes and in pain.

Accumulation of ß-Amyloid Leads to a Decrease in Lynx1 and Lypd6B Expression in the Hippocampus and Increased Expression of Proinflammatory Cytokines in the Hippocampus and Blood Serum.

Alzheimer's disease is a rapidly progressive neurodegenerative disease, the development of which is associated with the accumulation of ß-amyloid oligomers, dysfunction of the α7-nAChR nicotinic acetylcholine receptor, and activation of inflammation. Previously, we showed that the neuromodulator Lynx1, which belongs to the Ly6/uPAR family, competes with ß-amyloid(1-42) for binding to α7-nAChR. In this work, we studied the expression and localization of Ly6/uPAR family proteins in the hippocampus of 2xTg-AD transgenic mice that model AD and demonstrate increased amyloidosis in the brain. Using real-time PCR, we showed a decrease in the expression of the genes encoding Lynx1, Lypd6b, and the postsynaptic marker PSD95, as well as an increase in the expression of the TNFα gene in the hippocampus of 2xTg-AD mice. Histochemical analysis showed that, in the hippocampus of 2xTg-AD mice, Lynx1 does not colocalize with α7-nAChR, which can lead to the development of pathology when the receptor interacts with oligomeric ß-amyloid. In addition, in 2xTg-AD mice, activation of systemic inflammation was shown, which manifests itself in a decrease in the serum level of SLURP-1, a Ly6/uPAR family protein capable of regulating inflammatory processes, as well as in an increase in the content of proinflammatory cytokines TNFα and TNFß. Thus, α7-nAChR dysfunction and maintenance of the inflammatory microenvironment in the brain in Alzheimer's disease may be associated with a decrease in the expression of Ly6/uPAR family proteins that regulate α7-nAChR activity and inflammation.

New Three-Finger Protein from Starfish Asteria rubens Shares Structure and Pharmacology with Human Brain Neuromodulator Lynx2.

Three-finger proteins (TFPs) are small proteins with characteristic three-finger ß-structural fold stabilized by the system of conserved disulfide bonds. These proteins have been found in organisms from different taxonomic groups and perform various important regulatory functions or act as components of snake venoms. Recently, four TFPs (Lystars 1-4) with unknown function were identified in the coelomic fluid proteome of starfish A. rubens. Here we analyzed the genomes of A. rubens and A. planci starfishes and predicted additional five and six proteins containing three-finger domains, respectively. One of them, named Lystar5, is expressed in A. rubens coelomocytes and has sequence homology to the human brain neuromodulator Lynx2. The three-finger structure of Lystar5 close to the structure of Lynx2 was confirmed by NMR. Similar to Lynx2, Lystar5 negatively modulated α4ß2 nicotinic acetylcholine receptors (nAChRs) expressed in X. laevis oocytes. Incubation with Lystar5 decreased the expression of acetylcholine esterase and α4 and α7 nAChR subunits in the hippocampal neurons. In summary, for the first time we reported modulator of the cholinergic system in starfish.

Lynx1 Limits Dendritic Spine Turnover in the Adult Visual Cortex.

UNLABELLED: Dendritic spine turnover becomes limited in the adult cerebral cortex. Identification of specific aspects of spine dynamics that can be unmasked in adulthood and its regulatory molecular mechanisms could provide novel therapeutic targets for inducing plasticity at both the functional and structural levels for robust recovery from brain disorders and injuries in adults. Lynx1, an endogenous inhibitor of nicotinic acetylcholine receptors, was previously shown to increase its expression in adulthood and thus to limit functional ocular dominance plasticity in adult primary visual cortex (V1). However, the role of this "brake" on spine dynamics is not known. We examined the contribution of Lynx1 on dendritic spine turnover before and after monocular deprivation (MD) in adult V1 with chronic in vivo imaging using two-photon microscopy and determined the spine turnover rate of apical dendrites of layer 5 (L5) and L2/3 pyramidal neurons in adult V1 of Lynx1 knock-out (KO) mice. We found that the deletion of Lynx1 doubled the baseline spine turnover rate, suggesting that the spine dynamics in the adult cortex is actively limited by the presence of Lynx1. After MD, adult Lynx1-KO mice selectively exhibit higher rate of spine loss with no difference in gain rate in L5 neurons compared with control wild-type counterparts, revealing a key signature of spine dynamics associated with robust functional plasticity in adult V1. Overall, Lynx1 could be a promising therapeutic target to induce not only functional, but also structural plasticity at the level of spine dynamics in the adult brain. SIGNIFICANCE STATEMENT: Dendritic spine turnover becomes limited in the adult cortex. In mouse visual cortex, a premier model of experience-dependent plasticity, we found that the deletion of Lynx1, a nicotinic "brake" for functional plasticity, doubled the baseline spine turnover in adulthood, suggesting that the spine dynamics in the adult cortex is actively limited by Lynx1. After visual deprivation, spine loss, but not gain rate, remains higher in adult Lynx1 knock-out mice than in control wild-type mice, revealing a key signature of spine dynamics associated with robust functional plasticity. Lynx1 would be a promising target to induce not only functional, but also structural plasticity at the level of spine dynamics in adulthood.

The prototoxin LYPD6B modulates heteromeric α3ß4-containing nicotinic acetylcholine receptors, but not α7 homomers.

Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3ß4-, α3α5ß4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, ß4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For ß4-α3-α3-ß4-α3 and ß4-α3-ß4-α3-α3, LYPD6B decreased EC50 from 631 to 79 µM, reduced Imax by at least 59%, and decreased τ. For ß4-α3-α5D-ß4-α3 and ß4-α3-ß4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(ß4)2 and (α3)2(α5D)(ß4)2 and did not affect the properties of (α3)2(ß4)3, α7, or (α3)2(α5N)(ß4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(ß4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3ß4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.

Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1.

Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders. SIGNIFICANCE STATEMENT: Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction and ocular dominance plasticity in adult visual cortex. This is the first demonstration of adult brain to retain the intrinsic capacity to elevate tPA in an experience-dependent manner but is effectively limited by Lynx1. tPA-Lynx1 may potentially be a new candidate mechanism for interventions that were shown to activate plasticity in adult brain.

Water-soluble LYNX1 residues important for interaction with muscle-type and/or neuronal nicotinic receptors.

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

Selective targeting of α7 nicotinic acetylcholine receptor by synthetic peptide mimicking loop I of human SLURP-1 provides efficient and prolonged therapy of epidermoid carcinoma in vivo.

α7-Type nicotinic acetylcholine receptor (α7-nAChR) promotes the growth and metastasis of solid tumors. Secreted Ly6/uPAR-Related Protein 1 (SLURP-1) is a specific negative modulator of α7-nAChR produced by epithelial cells. Here, we investigated mechanisms of antiproliferative activity of recombinant SLURP-1 in epidermoid carcinoma A431 cells and activity of SLURP-1 and synthetic 21 a.a. peptide mimicking its loop I (Oncotag) in a xenograft mice model of epidermoid carcinoma. SLURP-1 inhibited the mitogenic pathways and transcription factors in A431 cells, and its antiproliferative activity depended on α7-nAChR. Intravenous treatment of mice with SLURP-1 or Oncotag for 10 days suppressed the tumor growth and metastasis and induced sustained changes in gene and microRNA expression in the tumors. Both SLURP-1 and Oncotag demonstrated no acute toxicity. Surprisingly, Oncotag led to a longer suppression of pro-oncogenic signaling and downregulated expression of pro-oncogenic miR-221 and upregulated expression of KLF4 protein responsible for control of cell differentiation. Affinity purification revealed SLURP-1 interactions with both α7-nAChR and EGFR and selective Oncotag interaction with α7-nAChR. Thus, the selective inhibition of α7-nAChRs by drugs based on Oncotag may be a promising strategy for cancer therapy.

Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome.

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.

Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice.

Lynx1 is a glycosylphosphatidylinositol (GPI)-linked protein shown to affect synaptic plasticity through modulation of nicotinic acetylcholine receptor (nAChR) subtypes in the brain. Because of this function and structural similarity to α-bungarotoxin, which binds muscle-specific nAChRs with high affinity, Lynx1 is a promising candidate for modulating nAChRs in skeletal muscles. However, little is known about the expression and roles of Lynx1 in skeletal muscles and neuromuscular junctions (NMJs). Here, we show that Lynx1 is expressed in skeletal muscles, increases during development, and concentrates at NMJs. We also demonstrate that Lynx1 interacts with muscle-specific nAChR subunits. Additionally, we present data indicating that Lynx1 deletion alters the response of skeletal muscles to cholinergic transmission and their contractile properties. Based on these findings, we asked if Lynx1 deletion affects developing and adult NMJs. Loss of Lynx1 had no effect on NMJs at postnatal day 9 (P9) and moderately increased their size at P21. Thus, Lynx1 plays a minor role in the structural development of NMJs. In 7- and 12-month-old mice lacking Lynx1, there is a marked increase in the incidence of NMJs with age- and disease-associated morphological alterations. The loss of Lynx1 also reduced the size of adult muscle fibers. Despite these effects, Lynx1 deletion did not alter the rate of NMJ reinnervation and stability following motor axon injury. These findings suggest that Lynx1 is not required during fast remodeling of the NMJ, as is the case during reformation following crushing of motor axons and development. Instead, these data indicate that the primary role of Lynx1 may be to maintain the structure and function of adult and aging NMJs.

SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer.

Secreted Ly6/uPAR-related protein 1 (SLURP-1) is a secreted Ly6/uPAR protein that negatively modulates the nicotinic acetylcholine receptor of α7 type (α7-nAChR), participating in control of cancer cell growth. Previously we showed, that a recombinant analogue of human SLURP-1 (rSLURP-1) diminishes the lung adenocarcinoma A549 cell proliferation and abolishes the nicotine-induced growth stimulation. Here, using multiplex immunoassay, we demonstrated a decrease in PTEN and mammalian target of rapamycin (mTOR) kinase phosphorylation in A549 cells upon the rSLURP-1 treatment pointing on down-regulation of the PI3K/AKT/mTOR signaling pathway. Decreased phosphorylation of the platelet-derived growth factor receptor type ß (PDGFRß) and arrest of the A549 cell cycle in the S and G2/M phases without apoptosis induction was also observed. Using a scratch migration assay, inhibition of A549 cell migration under the rSLURP-1 treatment was found. Affinity extraction demonstrated that rSLURP-1 in A549 cells forms a complex not only with α7-nAChR, but also with PDGFRα and epidermal growth factor receptor (EGFR), which are known to be involved in regulation of cancer cell growth and migration and are able to form a heterodimer. Knock-down of the genes encoding α7-nAChR, PDGFRα, and EGFR confirmed the involvement of these receptors in the anti-migration effect of SLURP-1. Thus, SLURP-1 can target the α7-nAChR complexes with PDGFRα and EGFR in the membrane of epithelial cells. Using chimeric proteins with grafted SLURP-1 loops we demonstrated that loop I is the principal active site responsible for the SLURP-1 interaction with α7-nAChR and its antiproliferative effect. Synthetic peptide mimicking the loop I cyclized by a disulfide bond inhibited ACh-evoked current at α7-nAChR, as well as A549 cell proliferation and migration. This synthetic peptide represents a promising prototype of new antitumor drug with the properties close to that of the native SLURP-1 protein.

Human Three-Finger Protein Lypd6 Is a Negative Modulator of the Cholinergic System in the Brain.

Lypd6 is a GPI-tethered protein from the Ly-6/uPAR family expressed in the brain. Lypd6 enhances the Wnt/ß-catenin signaling, although its action on nicotinic acetylcholine receptors (nAChRs) have been also proposed. To investigate a cholinergic activity of Lypd6, we studied a recombinant water-soluble variant of the human protein (ws-Lypd6) containing isolated "three-finger" LU-domain. Experiments at different nAChR subtypes expressed in Xenopus oocytes revealed the negative allosteric modulatory activity of ws-Lypd6. Ws-Lypd6 inhibited ACh-evoked currents at α3ß4- and α7-nAChRs with IC50 of ∼35 and 10 µM, respectively, and the maximal amplitude of inhibition of 30-50%. EC50 of ACh at α3ß4-nAChRs (∼30 µM) was not changed in the presence of 35 µM ws-Lypd6, while the maximal amplitude of ACh-evoked current was reduced by ∼20%. Ws-Lypd6 did not elicit currents through nAChRs in the absence of ACh. Application of 1 µM ws-Lypd6 significantly inhibited (up to ∼28%) choline-evoked current at α7-nAChRs in rat hippocampal slices. Similar to snake neurotoxin α-bungarotoxin, ws-Lypd6 suppressed the long-term potentiation (LTP) in mouse hippocampal slices. Colocalization of endogenous GPI-tethered Lypd6 with α3ß4- and α7-nAChRs was detected in primary cortical and hippocampal neurons. Ws-Lypd6 interaction with the extracellular domain of α7-nAChR was modeled using the ensemble protein-protein docking protocol. The interaction of all three Lypd6 loops ("fingers") with the entrance to the orthosteric ligand-binding site and the loop C of the primary receptor subunit was predicted. The results obtained allow us to consider Lypd6 as the endogenous negative modulator involved in the regulation of the cholinergic system in the brain.

Biophysical characterization of lynx-nicotinic receptor interactions using atomic force microscopy.

Nicotinic acetylcholine receptors (nAChRs) are broadly expressed in the central and peripheral nervous systems, playing essential roles in cholinergic neurotransmission. The lynx family proteins, a subset of the Ly6/uPAR superfamily expressed in multiple brain regions, have been shown to bind to nAChRs and modulate their function via allosteric regulation. The binding interactions between lynx and nAChRs, however, have not been systematically quantified and compared. In this work, we characterized the interactions between lynx1 or lynx2 and α3ß4- or α7-nAChRs using single-molecule atomic force microscopy (AFM). The AFM technique allows the quantification of the off-rate of lynx-nAChR binding and of the energetic barrier width between the bound state and transition state, providing a biophysical means to compare the selectivity of lynx proteins for nAChR subtypes. Results indicate that lynx1 has a marginal preference for α7- over α3ß4-nAChRs. Strikingly, lynx2 exhibits a two order of magnitude stronger affinity for α3ß4- compared to α7-nAChRs. Together, the AFM assay serves as a valuable tool for the biophysical characterization of lynx-nAChR binding affinities. Revealing the differential affinities of lynx proteins for nAChR subtypes will help elucidate how lynx regulates nAChR-dependent functions in the brain, including nicotine addiction and other critical pathways.

Nicotinic regulation of experience-dependent plasticity in visual cortex.

While the cholinergic neuromodulatory system and muscarinic acetylcholine receptors (AChRs) have been appreciated as permissive factors for developmental critical period plasticity in visual cortex, it was unknown why plasticity becomes limited after the critical period even in the presence of massive cholinergic projections to visual cortex. In this review we highlighted the recent progresses that started to shed light on the role of the nicotinic cholinergic neuromodulatory signaling on limiting juvenile form of plasticity in the adult brain. We introduce the Lynx family of proteins and Lynx1 as its representative, as endogenous proteins structurally similar to α-bungarotoxin with the ability to bind and modulate nAChRs to effectively regulate functional and structural plasticity. Remarkably, Lynx family members are expressed in distinct subpopulations of GABAergic interneurons, placing them in unique positions to potentially regulate the convergence of GABAergic and nicotinic neuromodulatory systems to regulate plasticity. Continuing studies of the potentially differential roles of Lynx family of proteins may further our understanding of the fundamentals of molecular and cell type-specific mechanisms of plasticity that we may be able to harness through nicotinic cholinergic signaling.

Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators.

Snake venom neurotoxins and lymphocyte antigen 6 (Ly6) proteins, most of the latter being membrane tethered by a glycosylphosphatidylinositol (GPI) anchor, have a variety of biological activities, but their three-finger (3F) folding combines them in one Ly6/neurotoxin family. Subsets of two groups, represented by α-neurotoxins and Lynx1, respectively, interact with nicotinic acetylcholine receptors (nAChR) and, hence, are of therapeutic interest for the treatment of neurodegenerative diseases, pain, and cancer. Information on the mechanisms of action and 3D structure of the binding sites, which is required for drug design, is available from the 3D structure of α-neurotoxin complexes with nAChR models. Here, I compare the structural and functional features of α-neurotoxins versus Lynx1 and its homologs to get a clearer picture of Lynx1-nAChR interactions that is necessary for fundamental science and practical applications.

Transcriptional regulation of SLURP2, a psoriasis-associated gene, is under control of IL-22 in the skin: A special reference to the nested gene LYNX1.

A novel nicotinic acetylcholine (ACh) receptor (nAChR)-mediated transduction pathway, regulating keratinocyte function, has been elucidated in studies of secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein (SLURP)-1 and -2. SLURPs are members of Ly6/neurotoxin superfamily (Ly6SF) of proteins containing the unique three-finger domain in their three-dimensional structure. Some endogenously expressed Ly6SF proteins (such as LYNX1, SLURP-1, and SLURP-2) modulate the function of nAChR, either as allosteric and/or orthosteric modulators, or as antagonists. Although the expression and functions of SLURP-1 and SLURP-2 in keratinocytes are well documented, the expression and the modes of action of LYNX1 in keratinocytes are unknown. Additionally, a particular hybrid transcript, LYNX1-SLURP2, which contains both LYNX1 and SLURP-2 sequences, with unknown function, has been reported. Furthermore, although SLURP2 is a gene strongly induced in psoriatic skin lesions, the mechanisms controlling SLURP2 expression are largely unknown. To better understand the function of nAChRs in keratinocytes, we investigated the expression profiles of LYNX1, LYNX1-SLURP-2, and SLURP-2 in keratinocytes under various inflammatory conditions. We found that keratinocytes express LYNX1 and SLURP2, but not LYNX1-SLURP2, at mRNA and protein levels. IL-22 treatment increased SLURP2 expression in keratinocytes, but this effect was completely abolished by IFN-γ. Furthermore, the IL-22-induced up-regulation of SLURP2 was completely suppressed by the inhibitor or siRNA for STAT3, a major transcriptional factor downstream of IL-22. These findings provide new insights into the nAChR-mediated regulatory mechanism of SLURP-2 expression in keratinocytes.