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.

Abnormal response to chronic social defeat stress and fear extinction in a mouse model of cholinergic dysregulation.

Cholinergic signaling is critical for an individual to react appropriately and adaptably to salient stimuli while navigating a complex environment. The cholinergic neurotransmitter system drives attention to salient stimuli, such as stressors, and aids in orchestrating the proper neural and behavioral response. Fine-tuned regulation of the cholinergic system has been linked to appropriate stress responses and subsequent mood regulation while dysregulation has been implicated in mood disorders. Among the multiple layers of regulation are cholinergic protein modulators. Here, we use validated models of experiential-based affective disorders to investigate differences in responses to stress in a genetic mouse model of cholinergic dysregulation based on the loss of protein modulator. The lynx2 nicotinic receptor modulatory protein provides negative cholinergic regulation within the amygdala, medial prefrontal cortex, and other brain regions. We discovered here that lynx2 knockout (KO) mice demonstrate an inability to update behavior with an inability to extinguish learned fear during a fear extinction test. We also observed, under an increased stress load following exposure to chronic social defeat stress (CSDS) paradigm, there was a unified resilience phenotype in lynx2KO mice, as opposed to the wild-type cohort which was split between resilience and susceptible phenotypes. Furthermore, we provide evidence for the functional role of α7 nicotinic receptor subtypes by phenotypic rescue with MLA or crossing with an α7 null mutant mouse (e.g. lynx2/α7 double KO mice). We demonstrate a direct physical interaction between lynx2 and α7 nAChR by co-immunoprecipitation of complexes from mouse BLA extracts. The genetic predisposition to heightened basal anxiety-like behavior and altered cholinergic signaling impairs individual behavior responses stressors. Together, these data indicate that the effects of social stress can be influenced by baseline genetic factors involved in anxiety regulation.

Analysis of Protein-Protein Interactions for Intermolecular Bond Prediction.

Protein-protein interactions often involve a complex system of intermolecular interactions between residues and atoms at the binding site. A comprehensive exploration of these interactions can help reveal key residues involved in protein-protein recognition that are not obvious using other protein analysis techniques. This paper presents and extends DiffBond, a novel method for identifying and classifying intermolecular bonds while applying standard definitions of bonds in chemical literature to explain protein interactions. DiffBond predicted intermolecular bonds from four protein complexes: Barnase-Barstar, Rap1a-raf, SMAD2-SMAD4, and a subset of complexes formed from three-finger toxins and nAChRs. Based on validation through manual literature search and through comparison of two protein complexes from the SKEMPI dataset, DiffBond was able to identify intermolecular ionic bonds and hydrogen bonds with high precision and recall, and identify salt bridges with high precision. DiffBond predictions on bond existence were also strongly correlated with observations of Gibbs free energy change and electrostatic complementarity in mutational experiments. DiffBond can be a powerful tool for predicting and characterizing influential residues in protein-protein interactions, and its predictions can support research in mutational experiments and drug design.

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.

DiffBond: A Method for Predicting Intermolecular Bond Formation.

Many tools that explore models of protein complexes are also able to analyze interactions between specific residues and atoms. A comprehensive exploration of these interactions can often uncover aspects of protein-protein recognition that are not obvious using other protein analysis techniques. This paper describes DiffBond, a novel method for searching for intermolecular interactions between protein complexes while differentiating between three different types of interaction: hydrogen bonds, ionic bonds, and salt bridges. DiffBond incorporates textbook definitions of these three interactions while contending with uncertainties that are inherent in computational models of interacting proteins. We used it to examine the barnase-barstar, Rap1a-raf, and Smad2-Smad4 complexes, as well as a subset of protein complexes formed between three-finger toxins and nAChRs. Based on electrostatic interactions established by previous experimental studies, DiffBond was able to identify ionic and hydrogen bonds with high precision and recall, and identify salt bridges with high precision. In combination with other electrostatic analysis methods, DiffBond can be a useful tool in helping predict influential amino acids in protein-protein interactions and characterizing the type of interaction.

Lynx1 prototoxins: critical accessory proteins of neuronal nicotinic acetylcholine receptors.

Nicotinic receptors of the cholinergic system are ligand-gated ion channels, responding to the excitatory neurotransmitter, acetylcholine, and the addictive component of tobacco, nicotine. They help to transduce salient information in the environment by activating specific neural circuitry in normal and disease states. While nicotinic receptors are promising neurological and neuropsychiatric disorder targets, they have fallen out of favor after several late-stage clinical failures. Targeting the complex of the nicotinic receptor, including lynx1 accessory proteins, could be the key to unlocking the intractable nAChR for therapeutic development. Lynx1 binds to the extracellular face of the nAChR and acts as a critical modulator, suppressing memory, learning, and plasticity. Lynx1 removal in animal models leads to memory and plasticity enhancements, some of which have therapeutic relevance for neuropsychiatric and neurological disease. A review of the lynx1 accessory modulator and its role in modulating neuronal nAChRs will be discussed.

Dynamics and Interactions of GPI-Linked lynx1 Protein with/without Nicotinic Acetylcholine Receptor in Membrane Bilayers.

Nicotinic acetylcholine receptors (nAChRs) participate in diverse biological processes, such as mood, learning, and addiction. Glycosylphosphatidylinositol-linked lynx1 is an allosteric modulator of nAChR function, including shifts in agonist sensitivity, reduced desensitization, and slower recovery from desensitization. This modulation is thought to be achieved by lynx1's interaction with nAChR subunits, particularly at the α4:α4 interface. In this study, we used molecular modeling and simulation to study the structure, dynamics, and interactions of lynx1 when bound to nAChRs, as well as unbound, monomeric lynx1 in membranes. Though lynx1 structures are similar in both states, its dynamics is more restricted in the bound state than in the unbound one. When bound, interactions between lynx1 and nAChR are observed to be maintained throughout the simulations. Of particular note, lynx1 demonstrates prolonged interactions with the receptor C-loop in one of the nAChR α4 subunits, a region important for agonist binding and possibly the transition between open/closed states. During interactions with lynx1, an α4 C-loop tends to be restricted in either a closed or open state, whereas the C-loop state transitions are more evident when lynx1 is unbound. Interestingly, the conformational change of the C-loop is stochastic, suggesting that lynx1 can influence nAChR (critical for its multimodal action), for instance, by shifting its agonist sensitivity and recovery from desensitization.

Neural layer self-assembly in geometrically confined rat and human 3D cultures.

Neurological disorders affect millions of Americans and this number is expected to rise with the aging population. Development of drugs to treat these disorders may be facilitated by improved in vitro models that faithfully reproduce salient features of the relevant brain regions. Current 3D culture methods face challenges with reliably reproducing microarchitectural features of brain morphology such as cortical or hippocampal layers. In this work, polydimethylsiloxane (PDMS) mini-wells were used to create low aspect ratio, adherent 3D constructs where neurons self-assemble into layers. Layer self-assembly was determined to depend on the size of the PDMS mini-well. Layer formation occurred in cultures composed of primary rat cortical neurons or human induced pluripotent stem cell-derived neurons and astrocytes and was robust and reproducible. Layered 3D constructs were found to have spontaneous neural activity characterized by long bursts similar to activity in the developing cortex. The responses of layered 3D cultures to drugs were more similar to in vivo data than those of 2D cultures. 3D constructs created with this method may be thus suitable as in vitro models for drug discovery for neurological disorders.

Lynx Prototoxins: Roles of Endogenous Mammalian Neurotoxin-Like Proteins in Modulating Nicotinic Acetylcholine Receptor Function to Influence Complex Biological Processes.

The cholinergic system modulates many biological functions, due to the widespread distribution of cholinergic neuronal terminals, and the diffuse release of its neurotransmitter, acetylcholine. Several layers of regulation help to refine and control the scope of this excitatory neurotransmitter system. One such regulatory mechanism is imparted through endogenous toxin-like proteins, prototoxins, which largely control the function of nicotinic receptors of the cholinergic system. Prototoxins and neurotoxins share the distinct three finger toxin fold, highly effective as a receptor binding protein, and the former are expressed in the mammalian brain, immune system, epithelium, etc. Prototoxins and elapid snake neurotoxins appear to be related through gene duplication and divergence from a common ancestral gene. Protein modulators can provide a graded response of the cholinergic system, and within the brain, stabilize neural circuitry through direct interaction with nicotinic receptors. Understanding the roles of each prototoxin (e.g., lynx1, lynx2/lypd1, PSCA, SLURP1, SLURP2, Lypd6, lypd6b, lypdg6e, PATE-M, PATE-B, etc.), their binding specificity and unique expression profile, has the potential to uncover many fascinating cholinergic-dependent mechanisms in the brain. Each family member can provide a spatially restricted level of control over nAChR function based on its expression in the brain. Due to the difficulty in the pharmacological targeting of nicotinic receptors in the brain as a result of widespread expression patterns and similarities in receptor sequences, unique interfaces between prototoxin and nicotinic receptor could provide more specific targeting than nicotinic receptors alone. As such, this family is intriguing from a long-term therapeutic perspective.

Augmenting the antinociceptive effects of nicotinic acetylcholine receptor activity through lynx1 modulation.

Neuronal nicotinic acetylcholine receptors (nAChRs) of the cholinergic system have been linked to antinociception, and therefore could be an alternative target for pain alleviation. nAChR activity has been shown to be regulated by the nicotinic modulator, lynx1, which forms stable complexes with nAChRs and has a negative allosteric action on their function. The objective in this study was to investigate the contribution of lynx1 to nicotine-mediated antinociception. Lynx1 contribution was investigated by mRNA expression analysis and electrophysiological responses to nicotine in the dorsal raphe nucleus (DRN), a part of the pain signaling pathway. In vivo antinociception was investigated in a test of nociception, the hot-plate analgesia assay with behavioral pharmacology. Lynx1/α4ß2 nAChR interactions were investigated using molecular dynamics computational modeling. Nicotine evoked responses in serotonergic and GABAergic neurons in the DRN are augmented in slices lacking lynx1 (lynx1KO). The antinociceptive effect of nicotine and epibatidine is enhanced in lynx1KO mice and blocked by mecamylamine and DHßE. Computer simulations predict preferential binding affinity of lynx1 to the α:α interface that exists in the stoichiometry of the low sensitivity (α4)3(ß2)2 nAChRs. Taken together, these data point to a role of lynx1 in mediating pain signaling in the DRN through preferential affinity to the low sensitivity α4ß2 nAChRs. This study suggests that lynx1 is a possible alternative avenue for nociceptive modulation outside of opioid-based strategies.

Deletion of lynx1 reduces the function of α6* nicotinic receptors.

The α6 nicotinic acetylcholine receptor (nAChR) subunit is an attractive drug target for treating nicotine addiction because it is present at limited sites in the brain including the reward pathway. Lynx1 modulates several nAChR subtypes; lynx1-nAChR interaction sites could possibly provide drug targets. We found that dopaminergic cells from the substantia nigra pars compacta (SNc) express lynx1 mRNA transcripts and, as assessed by co-immunoprecipitation, α6 receptors form stable complexes with lynx1 protein, although co-transfection with lynx1 did not affect nicotine-induced currents from cell lines transfected with α6 and ß2. To test whether lynx1 is important for the function of α6 nAChRs in vivo, we bred transgenic mice carrying a hypersensitive mutation in the α6 nAChR subunit (α6L9'S) with lynx1 knockout mice, providing a selective probe of the effects of lynx1 on α6* nAChRs. Lynx1 removal reduced the α6 component of nicotine-mediated rubidium efflux and dopamine (DA) release from synaptosomal preparations with no effect on numbers of α6ß2 binding sites, indicating that lynx1 is functionally important for α6* nAChR activity. No effects of lynx1 removal were detected on nicotine-induced currents in slices from SNc, suggesting that lynx1 affects presynaptic α6* nAChR function more than somatic function. In the absence of agonist, lynx1 removal did not alter DA release in dorsal striatum as measured by fast scan cyclic voltammetry. Lynx1 removal affected some behaviors, including a novel-environment assay and nicotine-stimulated locomotion. Trends in 24-hour home-cage behavior were also suggestive of an effect of lynx1 removal. Conditioned place preference for nicotine was not affected by lynx1 removal. The results show that some functional and behavioral aspects of α6-nAChRs are modulated by lynx1.

TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus.

TRPV1 is an ion channel activated by heat and pungent agents including capsaicin, and has been extensively studied in nociception of sensory neurons. However, the location and function of TRPV1 in the hippocampus is debated. We found that TRPV1 is expressed in oriens-lacunosum-moleculare (OLM) interneurons in the hippocampus, and promotes excitatory innervation. TRPV1 knockout mice have reduced glutamatergic innervation of OLM neurons. When activated by capsaicin, TRPV1 recruits more glutamatergic, but not GABAergic, terminals to OLM neurons in vitro. When TRPV1 is blocked, glutamatergic input to OLM neurons is dramatically reduced. Heterologous expression of TRPV1 also increases excitatory innervation. Moreover, TRPV1 knockouts have reduced Schaffer collateral LTP, which is rescued by activating OLM neurons with nicotine-via α2ß2-containing nicotinic receptors-to bypass innervation defects. Our results reveal a synaptogenic function of TRPV1 in a specific interneuron population in the hippocampus, where it is important for gating hippocampal plasticity.

Isoform-specific mechanisms of α3ß4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.

This study investigates-for the first time to our knowledge-the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3- and ß4-subunit-containing human nicotinic acetylcholine receptors (α3ß4*-nAChRs). Concatenated gene constructs were used to express precisely defined α3ß4*-nAChR isoforms (α3ß4)2ß4-, (α3ß4)2α3-, (α3ß4)2α5(398D)-, and (α3ß4)2α5(398N)-nAChR in Xenopus oocytes. In the presence or absence of lynx1, α3ß4*-nAChR agonist responses were recorded by using 2-electrode voltage clamp and single-channel electrophysiology, whereas radioimmunolabeling measured cell-surface expression. Lynx1 reduced (α3ß4)2ß4-nAChR function principally by lowering cell-surface expression, whereas single-channel effects were primarily responsible for reducing (α3ß4)2α3-nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3-fold reduction in the proportion of long bursts), and enhanced closed dwell times (3- to 6-fold increase)]. Alterations in both cell-surface expression and single-channel properties accounted for the reduction in (α3ß4)2α5-nAChR function that was mediated by lynx1. No effects were observed when α3ß4*-nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3ß4*-α5*-nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(-)-interface containing] by which prototoxins can modulate nAChR function.-George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform-specific mechanisms of α3ß4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.

Nicotinic receptor subtype-selective circuit patterns in the subthalamic nucleus.

The glutamatergic subthalamic nucleus (STN) exerts control over motor output through nuclei of the basal ganglia. High-frequency electrical stimuli in the STN effectively alleviate motor symptoms in movement disorders, and cholinergic stimulation boosts this effect. To gain knowledge about the mechanisms of cholinergic modulation in the STN, we studied cellular and circuit aspects of nicotinic acetylcholine receptors (nAChRs) in mouse STN. We discovered two largely divergent microcircuits in the STN; these are regulated in part by either α4ß2 or α7 nAChRs. STN neurons containing α4ß2 nAChRs (α4ß2 neurons) received more glutamatergic inputs, and preferentially innervated GABAergic neurons in the substantia nigra pars reticulata. In contrast, STN neurons containing α7 nAChRs (α7 neurons) received more GABAergic inputs, and preferentially innervated dopaminergic neurons in the substantia nigra pars compacta. Interestingly, local electrical stimuli excited a majority (79%) of α4ß2 neurons but exerted strong inhibition in 58% of α7 neurons, indicating an additional diversity of STN neurons: responses to electrical stimulation. Chronic exposure to nicotine selectively affects α4ß2 nAChRs in STN: this treatment increased the number of α4ß2 neurons, upregulated α4-containing nAChR number and sensitivity, and enhanced the basal firing rate of α4ß2 neurons both ex vivo and in vivo. Thus, chronic nicotine enhances the function of the microcircuit involving α4ß2 nAChRs. This indicates chronic exposure to nicotinic agonist as a potential pharmacological intervention to alter selectively the balance between these two microcircuits, and may provide a means to inhibit substantia nigra dopaminergic neurons.

Lynx1 shifts α4ß2 nicotinic receptor subunit stoichiometry by affecting assembly in the endoplasmic reticulum.

Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4ß2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and ß2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)3(ß2)2 pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein.

Lynx1 supports neuronal health in the mouse dorsal striatum during aging: an ultrastructural investigation.

Nicotinic acetylcholine receptors have been shown to participate in neuroprotection in the aging brain. Lynx protein modulators dampen the activity of the cholinergic system through direct interaction with nicotinic receptors. Although lynx1 null mutant mice exhibit augmented learning and plasticity, they also exhibit macroscopic vacuolation in the dorsal striatum as they age, detectable at the optical microscope level. Despite the relevance of the lynx1 gene to brain function, little is known about the cellular ultrastructure of these age-related changes. In this study, we assessed degeneration in the dorsal striatum in 1-, 3-, 7-, and 13-month-old mice, using optical and transmission electron microscopy. We observed a loss of nerve fibers, a breakdown in nerve fiber bundles, and a loss of neuronal nuclei in the 13-month-old lynx1 null striatum. At higher magnification, these nerve fibers displayed intracellular vacuoles and disordered myelin sheaths. Few or none of these morphological alterations were present in younger lynx1 null mutant mice or in heterozygous lynx1 null mutant mice at any age. These data indicate that neuronal health can be maintained by titrating lynx1 dosage and that the lynx1 gene may participate in a trade-off between neuroprotection and augmented learning.

Enhancement in motor learning through genetic manipulation of the Lynx1 gene.

The cholinergic system is a neuromodulatory neurotransmitter system involved in a variety of brain processes, including learning and memory, attention, and motor processes, among others. The influence of nicotinic acetylcholine receptors of the cholinergic system are moderated by lynx proteins, which are GPI-anchored membrane proteins forming tight associations with nicotinic receptors. Previous studies indicate lynx1 inhibits nicotinic receptor function and limits neuronal plasticity. We sought to investigate the mechanism of action of lynx1 on nicotinic receptor function, through the generation of lynx mouse models, expressing a soluble version of lynx and comparing results to the full length overexpression. Using rotarod as a test for motor learning, we found that expressing a secreted variant of lynx leads to motor learning enhancements whereas overexpression of full-length lynx had no effect. Further, adult lynx1KO mice demonstrated comparable motor learning enhancements as the soluble transgenic lines, whereas previously, aged lynx1KO mice showed performance augmentation only with nicotine treatment. From this we conclude the motor learning is more sensitive to loss of lynx function, and that the GPI anchor plays a role in the normal function of the lynx protein. In addition, our data suggests that the lynx gene plays a modulatory role in the brain during aging, and that a soluble version of lynx has potential as a tool for adjusting cholinergic-dependent plasticity and learning mechanisms in the brain.

Optimizing cholinergic tone through lynx modulators of nicotinic receptors: implications for plasticity and nicotine addiction.

The cholinergic system underlies both adaptive (learning and memory) and nonadaptive (addiction and dependency) behavioral changes through its ability to shape and regulate plasticity. Protein modulators such as lynx family members can fine tune the activity of the cholinergic system and contribute to the graded response of the cholinergic system, stabilizing neural circuitry through direct interaction with nicotinic receptors. Release of this molecular brake can unmask cholinergic-dependent mechanisms in the brain. Lynx proteins have the potential to provide top-down control over plasticity mechanisms, including addictive propensity. If this is indeed the case, then, what regulates the regulator? Transcriptional changes of lynx genes in response to pharmacological, physiological, and pathological alterations are explored in this review.