Orthogonal array formation by human aquaporin-4: examination of neuromyelitis optica-associated aquaporin-4 polymorphisms.

Pathogenic autoantibodies target aquaporin-4 (AQP4) water channels in individuals with neuromyelitis optica (NMO). Recently, allelic mutations were reported at residue 19 of AQP4 in three cases of NMO, and it was suggested that polymorphisms may influence disease by altering AQP4 supramolecular assembly into orthogonal arrays of particles (OAPs). We analyzed the determinants of OAP formation by human AQP4 to investigate the possible role of polymorphisms in NMO pathogenesis. NMO-associated mutations R19I and R19T in AQP4 did not affect OAP assembly, palmitoylation-dependent regulation of assembly, or NMO autoantibody binding. Residue-19 polymorphisms in AQP4 are thus unlikely to be disease relevant.

Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays.

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.

Evaluation of a pharmacokinetic hypothesis for reduced locomotor stimulation from methamphetamine and cocaine in adolescent versus adult male C57BL/6J mice.

RATIONALE: Adolescent mice display reduced locomotor stimulation to cocaine and amphetamine compared to adults, but the mechanisms are not known. OBJECTIVES: The primary aim of the current study is to test a possible pharmacokinetic explanation for the attenuated locomotor stimulation seen in adolescents. A secondary aim is to extend the current literature for acute methamphetamine in adolescents. MATERIALS AND METHODS: Male, adolescent (PN 30-35) and adult (PN 69-74) C57BL/6J mice were administered an intraperitoneal injection of cocaine (5, 15, 30 mg/kg) or methamphetamine (1, 2, 4 mg/kg) and euthanized 5, 10, 15, 30, 60, 120, or 240 min later. Home cage locomotor activity was recorded by video tracking, and drug concentration levels in brain and blood from the infraorbital sinus were measured using liquid chromatography combined with mass spectroscopy. RESULTS: Both methamphetamine and cocaine increased locomotor activity in a dose-response fashion, but the magnitude of the increase was less in adolescents than adults. Concentration of methamphetamine in the brain was similar between ages across time points. Concentration of cocaine in the brain was significantly higher in adolescents than adults at 5 min, but similar at all other time points. CONCLUSIONS: Results suggest pharmacokinetics may make a small contribution to differential stimulation between adolescents and adult mice, but are unlikely the only factor. Developmental differences within the brain that effect pharmacodynamic properties of psychostimulants (e.g., number of receptor or transporters) represent alternatives.

Acute effects of acamprosate and MPEP on ethanol Drinking-in-the-Dark in male C57BL/6J mice.

BACKGROUND: Recently, a simple procedure in mice, Drinking-in-the-Dark (DID), was hypothesized to have value for medication development for human alcoholism. In DID, mice are offered intermittent, limited access to ethanol over a series of days during the dark phase that results in rapid drinking to intoxication in predisposed genotypes. METHODS: We measured the effects of acamprosate or MPEP, metabotropic glutamate 5 receptor (mGluR5) antagonist, on intake of 20% ethanol, plain tap water or 10% sugar water using the DID procedure in male C57BL/6J mice. RESULTS: Acamprosate (100, 200, 300, or 400 mg/kg) dose dependently decreased ethanol drinking with 300 mg/kg reducing ethanol intake by approximately 20% without affecting intake of plain water or 10% sugar water. MPEP (1, 3, 5, 10, 20, or 40 mg/kg) was more potent than acamprosate with 20 mg/kg reducing ethanol intake by approximately 20% and for longer duration without affecting intake of plain water or 10% sugar water. CONCLUSIONS: These results support the hypothesis that mGluR5 signaling plays a role in excessive ethanol intake in DID and suggest DID may have value for screening novel compounds that reduce overactive glutamate signaling for potential pharmaceutical treatment of excessive ethanol drinking behavior.