Reconfiguration of dominant coupling modes in mild traumatic brain injury mediated by δ-band activity: a resting state MEG study

During the last few years, rich-club (RC) organization has been studied as a possible brain-connectivity organization model for large-scale brain networks. At the same time, empirical and simulated data of neurophysiological models have demonstrated the significant role of intra-frequency and inter-frequency coupling among distinct brain areas. The current study investigates further the importance of these couplings using recordings of resting-state magnetoencephalographic activity obtained from 30 mild traumatic brain injury (mTBI) subjects and 50 healthy controls. Intra-frequency and inter-frequency coupling modes are incorporated in a single graph to detect group differences within individual rich-club subnetworks (type I networks) and networks connecting RC nodes with the rest of the nodes (type II networks). Our results show a higher probability of inter-frequency coupling for (δ–γ1), (δ–γ2), (θ–β), (θ–γ2), (α–γ2), (γ1–γ2) and intra-frequency coupling for (γ1–γ1) and (δ–δ) for both type I and type II networks in the mTBI group. Additionally, mTBI and control subjects can be correctly classified with high accuracy (98.6%), whereas a general linear regression model can effectively predict the subject group using the ratio of type I and type II coupling in the (δ, θ), (δ, β), (δ, γ1), and (δ, γ2) frequency pairs. These findings support the presence of an RC organization simultaneously with dominant frequency interactions within a single functional graph. Our results demonstrate a hyperactivation of intrinsic RC networks in mTBI subjects compared to controls, which can be seen as a plausible compensatory mechanism for alternative frequency-dependent routes of information flow in mTBI subjects

Improving the detection of mtbi via complexity analysis in resting - state magnetoencephalography

Diagnosis of mild Traumatic Brain Injury (mTBI) is difficult due to the variability of obvious brain lesions using imaging scans. A promising tool for exploring potential biomarkers for mTBI is magnetoencephalography which has the advantage of high spatial and temporal resolution. By adopting proper analytic tools from the field of symbolic dynamics like Lempel-Ziv complexity, we can objectively characterize neural network alterations compared to healthy control by enumerating the different patterns of a symbolic sequence. This procedure oversimplifies the rich information of brain activity captured via MEG. For that reason, we adopted neural-gas algorithm which can transform a time series into more than two symbols by learning brain dynamics with a small reconstructed error. The proposed analysis was applied to recordings of 30 mTBI patients and 50 normal controls in δ frequency band. Our results demonstrated that mTBI patients could be separated from normal controls with more than 97% classification accuracy based on high complexity regions corresponding to right frontal areas. In addition, a reverse relation between complexity and transition rate was demonstrated for both groups. These findings indicate that symbolic complexity could have a significant predictive value in the development of reliable biomarkers to help with the early detection of mTBI

Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data

Altered cross-frequency coupling in resting-state MEG after mild traumatic brain injury

Cross-frequency coupling (CFC) is thought to represent a basic mechanism of functional integration of neural networks across distant brain regions. In this study, we analyzed CFC profiles from resting state Magnetoencephalographic (MEG) recordings obtained from 30 mild traumatic brain injury (mTBI) patients and 50 controls. We used mutual information (MI) to quantify the phase-to-amplitude coupling (PAC) of activity among the recording sensors in six nonoverlapping frequency bands. After forming the CFC-based functional connectivity graphs, we employed a tensor representation and tensor subspace analysis to identify the optimal set of features for subject classification as mTBI or control. Our results showed that controls formed a dense network of stronger local and global connections indicating higher functional integration compared to mTBI patients. Furthermore, mTBI patients could be separated from controls with more than 90% classification accuracy. These findings indicate that analysis of brain networks computed from resting-state MEG with PAC and tensorial representation of connectivity profiles may provide a valuable biomarker for the diagnosis of mTBI

Expression and structural studies of fragments of nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of pentameric ligand-gated ion channels (LGICs), also including serotonin (5-HT3), glycine and γ- aminobutyric acid receptors (GABAA and GABAC). Each nAChR subunit consists of an Nterminal extracellular domain (ECD), harbouring the signature Cys-loop, four transmembrane α-helices and a small cytoplasmic loop. nAChRs are classified into muscle and neuronal types. Muscle-type nAChRs are found in fish electric organs and at the vertebrate neuromuscular junctions, where they mediate neuromuscular transmission. They form heteropentamers with a stoichiometry (α1)2β1γδ or (α1)2β1εδ in adult mammalian nAChR and bear two ligand-binding sites formed between α1- and γ(ε)- or δ-ECDs. The α1-ECD hosts the main immunogenic region (MIR), against which a large number of anti-nAChR antibodies are directed in the autoimmune disease, myasthenia gravis. Neuronal nAChRs are widely distributed in the central and peripheral nervous system and play key roles in neuron-neuron interactions. They exist either as heteropentamers of 2-3 α subunits (subtypes α2-6) plus 2-3 β subunits (β2-4) or as homopentamers. α7 is the only human neuronal subunit known to form a homopentamer with five ligand-binding sites between its ECDs. Regarding the atomic structure of nAChR, four major breakthroughs have been achieved so far: A. The X-ray crystal structure of the homologous to nAChR α-ECDs (25% sequence identity with α7), molluscan homopentameric acetylcholine-binding protein (AChBP) and its complexes with various cholinergic ligands, which approached the structure of the nAChR ligand-binding site in atomic detail for the first time. Β. Τhe 4A electron microscopy (EM) structure of the Torpedo nAChR, which revealed the architecture of the muscle-type nAChR-ECDs and the fivefold symmetry of the receptor. Nevertheless, given the relatively low-resolution, no atomic details for the ligand-binding site could be observed. C. The high-resolution X-ray crystal structure of the complex of mouse muscle α1-ECD with the nAChR antagonist α-bungarotoxin (α-bgtx), which revealed atomic details for the MIR epitope, the loops A, B and C, forming the principal side of the nAChR ligandbinding pocket and the Cys-loop. However, since the structure of mouse α1-ECD is a monomer, the nAChR ligand-binding site is still missing. D. The high-resolution X-ray crystal structure of a prokaryotic LGIC (ELIC protein) considered to be the ancestor of eukaryotic LGICS, including the nAChR, which revealed the core structure of the LGICs. Apparently, it still remains essential to obtain the structure of a pentameric nAChR-ECD in high-resolution, so as to look deep into the details of the complete nAChR ligand-binding pockets. Neuronal α7 nAChR is a good candidate for achieving this goal, as it forms homopentamers. Furthermore, since α7 nAChR is implicated in neurological diseases and disorders (Alzheimer’s, Parkinson’s, epilepsy, schizophrenia, etc), elucidation of its structure will also lead to the rational drug-design towards these diseases. Furthermore, since the α7-ECD is of the main pharmacological interest as this bears the ligand-binding sites, it seems more realistic to perform crystallization trials on this domain, rather than on the intact α7 nAChR, due to the large hydrophobic transmembrane domains of the latter. Our laboratory has previously expressed the wild-type and a double mutant of human α7-ECD in yeast P. pastoris, with the aim to proceed to their detailed structural analysis. However, the wild-type was expressed in the form of microaggregates and oligomers larger than the expected pentamers, whereas the double mutant, carrying the mutation Cys116Ser and the replacement of its Cys-loop by the more hydrophilic AChBP Cys-loop, appeared to be more soluble than the wild-type and capable of binding α-bgtx with an increased affinity, relatively close to that of the native α7 nAChR. However, this mutant was still aggregationprone to some extent, thus leading to unsuccessful crystallization trials. Therefore, in the present study, we based on the model of human α7-ECD constructed using as templates the X-ray crystal structure of L-AChBP and the electron microscopy structure of the Torpedo nAChR α1-ECD, and introduced several mutations in α7-ECD so as to enhance both its solubility and assembly to pentamers. The hydrophobic amino acid residues found exposed to the environment of α7-ECD model were mutated to less hydrophobic ones, with the aim to reduce the considerably high hydrophobicity of α7-ECD molecules, while various residues facing the interface between two adjacent α7-ECD protomers were mutated to larger or charged residues, with the aim to introduce additional hydrogen or electrostatic bonds with facing residues of the adjacent protomer.Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) ανήκουν στην υπερ-οικογένεια των πενταμερών ιοντικών καναλιών (LGICs), που περιλαμβάνει τους υποδοχείς σεροτονίνης (5-HT3), γλυκίνης (GlyR) και γ-αμινοβουτυρικού οξέος (GABAA, GABAB). Κάθε υπομονάδα του nAChR αποτελείται από μία αμινο-τελική εξωκυτταρική περιοχή (ΕΚΠ), στην οποία βρίσκεται η χαρακτηριστική Cys θηλιά της υπερ-οικογένειας, από τέσσερεις διαμεμβρανικές α-έλικες και από μία κυτταροπλασματική περιοχή. Οι nAChRs διακρίνονται σε μυϊκούς και νευρικούς. Οι μυϊκοί nAChRs βρίσκονται στα ηλεκτρικά όργανα ιχθύων Torpedo sp. και στις νευρομυϊκές συνάψεις σπονδυλωτών, όπου μεταβιβάζουν τις νευρικές ώσεις στους μείς. Σχηματίζουν ετεροπενταμερή με στοιχειομετρία υπομονάδων (α1)2β1γδ ή (α1)2β1εδ στα ενήλικα άτομα θηλαστικών, με δύο θέσεις πρόσδεσης χολινεργικών προσδετών μεταξύ των α1-ΕΚΠ και της γ(ε) και δ-ΕΚΠ. Στην α1-ΕΚΠ εδράζει επίσης η κύρια ανοσογόνος περιοχή (MIR), έναντι της οποίας κατευθύνονται αντι-nAChR αντισώματα στην περίπτωση της βαριάς μυασθένειας. Οι νευρικοί nAChRs βρίσκονται στο κεντρικό και περιφερικό νευρικό σύστημα, όπου διαβιβάζουν τις νευρικές ώσεις. Σχηματίζουν είτε ετεροπενταμερή, μεταξύ 2-3 α-υπομονάδων (υπότυποι α2-6) και 2-3 β-υπομονάδων (υπότυποι β2-4), ή ομοπενταμερή. Η α7 είναι η μόνη γνωστή υπομονάδα του νευρικού nAChR που σχηματίζει ομοπενταμερή μόρια στον άνθρωπο, με πέντε θέσεις πρόσδεσης χολινεργικών υποκαταστατών. Μέχρι στιγμής, έχουν πραγματοποιηθεί τέσσερα σημαντικά επιτεύγματα, όσον αφορά την κατανόηση της δομής των nAChRs: Α. Η λύση της δομής, με κρυσταλλογραφία ακτίνων-Χ, της ομόλογης προς τα ΕΚΠ τμήματα των α υπομονάδων του nAChR (25% αμινοξική ταύτιση με την α7), ομοπενταμερούς πρωτεΐνης δέσμευσης της ACh γαστεροπόδων (AChBP), καθώς και των δομών της με διάφορους χολινεργικούς προσδέτες. Προσεγγίστηκε έτσι για πρώτη φορά η διαμόρφωση των nAChR-θέσεων πρόσδεσης χολινεργικών υποκαταστατών. Β. Η λύση της δομής, με ηλεκτρονική μικροσκοπία, του Torpedo nAChR, η οποία αποκάλυψε την δευτεροταγή και τριτοταγή δομή του nAChR. Εντούτοις, λόγω της περιορισμένης ευκρίνειας (4 A), δεν αποκάλυψε τη συγκρότηση των θέσεων πρόσδεσης της ACh σε ατομικό επίπεδο. Γ. Η λύση της δομής σε υψηλή ευκρίνεια (1,94 A) του συμπλόκου της α1-ΕΚΠ επίμυος με τον nAChR- ανταγωνιστή α-μπουγκαροτοξίνη (α-bgtx). Η λυση αυτής της δομής αποκάλυψε σε ατομικό επίπεδο την MIR περιοχή, τις θηλιές Α, B και C της κυρίως πλευράς των θέσεων πρόσδεσης χολινεργικών προσδετών και τη χαρακτηριστική Cys θηλιά. Δεδομένου όμως, ότι η δομή της α1-ΕΚΠ αναπαριστά ένα μονομερές, η δομή μίας πλήρως διαμορφωμένης θέσης πρόσδεσης εξακολουθεί να απουσιάζει. Δ. Η λύση της δομής, με κρυσταλλογραφία ακτίνων-Χ, μίας προκαρυωτικής LGIC πρωτεΐνης (ELIC), η οποία θεωρείται μάλιστα πως αποτελεί τον πρόγονο όλων των ευκαρυωτικών LGICs, συμπεριλαμαβανομένου του nAChR, με την οποία αποκαλύφθηκε ο βασικός σκελετός της δομής των LGICs. Είναι φανερό, ότι παρόλα αυτά τα επιτεύγματα, απουσιάζει ακόμη η λύση της δομής ενός πενταμερούς nAChR. Η νευρική α7 υπομονάδα του nAChR είναι μία καλή υποψήφια για την επίτευξη αυτού του στόχου, αφού σχηματίζει πενταμερή in vivo. Επιπλέον, αυτή εμπλέκεται σε ένα μεγάλο αριθμό νευρικών παθήσεων (Alzheimer, Parkinson, επιληψία, σχιζοφρένεια, κλπ), και η λύση της δομής της θα οδηγήσει και στο σχεδιασμό θεραπευτικών προσεγγίσεων έναντι αυτών των ασθενειών. Μάλιστα, εφ’όσον στην α7- ΕΚΠ εδράζουν οι θέσεις πρόσδεσης χολινεργικών υποκαταστατών, είναι πιο ρεαλιστική η προσπάθεια κρυστάλλωσης αυτής της περιοχής αντί ολόκληρου του α7 nAChR, αφού οι εκτενείς υδρόφοβες διαμεμβρανικές περιοχές του τελευταίου περιορίζουν τη διαλυτότητά του. Στο παρελθόν είχαμε εκφράσει στο Εργαστήριό μας την ανθρώπινη α7-EKΠ αγρίου τύπου και ένα διπλό μετάλλαγμα αυτής, σε κύτταρα P. pastoris, με στόχο μελέτη της δομής τους. Τα μόρια αγρίου τύπου, βρέθηκαν όμως να είναι αρκετά υδρόφοβα, αφού εκφράσθηκαν ως συσσωματώματα πολύ υψηλού μοριακού βάρους (ΜΒ) και ως ολιγομερή μεγαλύτερα από πενταμερή. Τα μόρια του διπλού μεταλλάγματος, στα οποία ολόκληρη η Cys θηλιά του α7 nAChR αντικαταστάθηκε από την πιο υδρόφιλη Cys θηλιά της AChBP σε συνδυασμό με την μετάλλαξη Cys116Ser, εμφάνισαν μεν αυξημένη διαλυτότητα και ικανότητα πρόσδεσης α-bgtx, διατήρησαν όμως δε ένα σημαντικό βαθμό δημιουργίας συσσωματωμάτων υψηλού ΜΒ, οδηγώντας σε αποτυχείς προσπάθειες κρυστάλλωσής τους

Structural Insights into the Role of β3 nAChR Subunit in the Activation of Nicotinic Receptors

The β3 subunit of nicotinic acetylcholine receptors (nAChRs) participates in heteropentameric assemblies with some α and other β neuronal subunits forming a plethora of various subtypes, differing in their electrophysiological and pharmacological properties. While β3 has for several years been considered an accessory subunit without direct participation in the formation of functional binding sites, recent electrophysiology data have disputed this notion and indicated the presence of a functional (+) side on the extracellular domain (ECD) of β3. In this study, we present the 2.4 Å resolution crystal structure of the monomeric β3 ECD, which revealed rather distinctive loop C features as compared to those of α nAChR subunits, leading to intramolecular stereochemical hindrance of the binding site cavity. Vigorous molecular dynamics simulations in the context of full length pentameric β3-containing nAChRs, while not excluding the possibility of a β3 (+) binding site, demonstrate that this site cannot efficiently accommodate the agonist nicotine. From the structural perspective, our results endorse the accessory rather than functional role of the β3 nAChR subunit, in accordance with earlier functional studies on β3-containing nAChRs

Structural Insights into the Role of β3 nAChR Subunit in the Activation of Nicotinic Receptors

The β3 subunit of nicotinic acetylcholine receptors (nAChRs) participates in heteropentameric assemblies with some α and other β neuronal subunits forming a plethora of various subtypes, differing in their electrophysiological and pharmacological properties. While β3 has for several years been considered an accessory subunit without direct participation in the formation of functional binding sites, recent electrophysiology data have disputed this notion and indicated the presence of a functional (+) side on the extracellular domain (ECD) of β3. In this study, we present the 2.4 Å resolution crystal structure of the monomeric β3 ECD, which revealed rather distinctive loop C features as compared to those of α nAChR subunits, leading to intramolecular stereochemical hindrance of the binding site cavity. Vigorous molecular dynamics simulations in the context of full length pentameric β3-containing nAChRs, while not excluding the possibility of a β3 (+) binding site, demonstrate that this site cannot efficiently accommodate the agonist nicotine. From the structural perspective, our results endorse the accessory rather than functional role of the β3 nAChR subunit, in accordance with earlier functional studies on β3-containing nAChRs

Molecular interaction of α-conotoxin RgIA with the rat α9α10 nicotinic acetylcholine receptors

The α9α10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that α-conotoxin (α-CTx) RgIA, one of the few α9α10 selective ligands identified to date, is 300-fold less potent on human versus rat α9α10 nAChR. This species difference was conferred by only one residue in the (-), rather than (+), binding region of the α9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with α-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the α9(+) face and two residues on the α10(-) face of the α9α10 nAChR. However, mutations of these residues had little or no effect on toxin block of the α9α10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (α10 E197, P200 and α9 T61, D121) resulted in 19- to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human α9 nAChR, we modeled the rat α9α10 ECD and its complexes with α-CTx RgIA and acetylcholine. Our data support the interaction of α-CTx RgIA at the α10/α9 rather than the α9/α10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.</p

Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies