Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders

BackgroundNeuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in “orphan” AES cases.MethodsThe study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 “control” patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR–transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies’ binding to rat brain tissue.ResultsThree patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient‐derived serum anti‐nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti‐nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti‐nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis.ConclusionThis study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Sex-stratified Genome-wide Association Studies Including 270,000 Individuals Show Sexual Dimorphism in Genetic Loci for Anthropometric Traits

Peer reviewe

Efficient Expression of Functional (α6β2)₂β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

<div><p>Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)₂β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)₂β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.</p></div

Concentration/response curves for constructs that resulted in significant amounts of functional AChRs.

<p>Full agonist (ACh) and partial agonists (cytisine, nicotine, and varenicline) were used on (α6β2)₂β3 AChRs. Each point is the average response of at least 5 oocytes. Arrows indicate construct <b>5</b>, that behaves like construct <b>1</b>, and constructs <b>6</b> and <b>9</b> that are divergent.</p

Assembly of (α6β2)₂β3 AChRs constructs evaluated by sucrose sedimentation velocity gradient analysis.

<p>After centrifugation, gradient fractions were immunoisolated on microwells coated with mAb 295 to β2 to isolate α6β2β3 AChRs prior to labeling with ³H epibatidine. Properly assembled mature (α6β2)₂β3 AChRs sediment between the two internal standards, 9S monomer and 13S dimer of <i>Torpedo californica</i> AChRs. Peaks on the left of dimers in the gradient indicate multimers or aggregates of AChRs, while peaks on the right of monomers represent partially assembled AChRs. A) Expression of pentameric concatamer construct <b>1</b> (β3−α6−β2−α6−β2) resulted in a high proportion of mature AChRs, as expected (16). B) Expression of construct <b>2</b> (α6+β2+β3) resulted in a high proportion of aggregates and partially assembled AChRs and a very low proportion of mature AChRs, as expected (12). C) Expression of construct <b>3</b> (α6_211L+β2+β3) resulted in a large proportion of mature AChRs. D) Expression of construct <b>4</b> (α6_α3cyt+β2+β3) resulted in mature AChRs but also partially assembled AChRs and both large and very large aggregates. E) Expression of construct <b>5</b> (α6_211L,α3cyt+β2+β3) showed mature AChRs and some aggregates. F) Expresion of construct <b>6</b> (α6_α3cyt-C+β2+β3) showed a high proportion of mature AChRs and few aggregates.</p

Illustration of (α6β2)₂β3 AChR constructs.

<p>A) Diagrammatic representation of an AChR subunit. B) Diagrammatic representation of two AChR subunits joined by a linker. Direction of the linker is indicated by arrows. C) Representation of α6 and α3 sequences used in the α6/α3 chimeras studied. D) Representation of (α6β2)₂β3 AChRs assembled from the various constructs. Agonist binding sites are shown as solid triangles between two subunits. The number and nomenclature for each construct depicted here are used in the following data figures.</p

β3 incorporation in (α6β2)₂β3 AChR constructs.

<p>Microwells were coated with mAb 295 to bind AChRs containing β2 or mAb 210 to bind AChRs containing β3. Detergent-solubilized AChRs were added to the wells. Bound AChRs were then assayed by binding of ³H epibatidine.</p

Kinetics of responses to increasing concentrations of ACh by constructs 1 and 5.

<p>A) Concatamer <b>1</b> (β3−α6−β2−α6−β2) responds more rapidly with greater currents and more extensive desensitization at higher ACh concentrations. B) Construct <b>5</b> (α6_211L,α3cyt+β2+β3) response kinetics to ACh are similar, but amplitudes of responses are much larger.</p