Supramolecular aggregation of aquaporin-4 is different in muscle and brain: correlation with tissue susceptibility in neuromyelitis optica

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by autoantibodies (NMO-IgG) against the water channel aquaporin-4 (AQP4). Though AQP4 is also expressed outside the CNS, for example in skeletal muscle, patients with NMO generally do not show clinical/diagnostic evidence of skeletal muscle damage. Here, we have evaluated whether AQP4 supramolecular organization is at the basis of the different tissue susceptibility. Using immunofluorescence we found that while the sera of our cohort of patients with NMO gave typical perivascular staining in the CNS, they were largely negative in the skeletal muscle. This conclusion was obtained using human, rat and mouse skeletal muscle including the AQP4-KO mouse. A biochemical analysis using a new size exclusion chromatography approach for AQP4 suprastructure fractionation revealed substantial differences in supramolecular AQP4 assemblies and isoform abundance between brain and skeletal muscle matching a lower binding affinity of NMO-IgG to muscle compared to the brain. Super-resolution microscopy analysis with g-STED revealed different AQP4 organization in native tissues, while in the brain perivascular astrocyte endfoot membrane AQP4 was mainly organized in large interconnected and raft-like clusters, in the sarcolemma of fast-twitch fibres AQP4 aggregates often appeared as small, relatively isolated linear entities. In conclusion, our results provide evidence that AQP4 supramolecular structure is different in brain and skeletal muscle, which is likely to result in different tissues susceptibility to the NMO disease

Development of an aquaporin-4 orthogonal array of particle-based ELISA for neuromyelitis optica autoantibodies detection

Serological markers of Nuromyelitis Optica (NMO), an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG) are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs). Many tests have been developed to detect AQP4- IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA), which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91%) compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used. © 2015 Pisani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica Autoantibodies

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp69) of TM2 for NMO-IgG epitope assembly. Mutation of Asp69to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp69mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D69H mutation has the effect of altering the structural rear-rangements of extracellular loop A. In conclusion, Asp69is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial-mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells' infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment

Detection limits of commercial ELISA, OAP-ELISA and CBA.

<p>(A) Positive controls (CI, CII), High- (H), Low- (L) titer NMO sera and MS sera were tested using limiting dilution analysis by commercial ELISA. (B) MS and H and L NMO sera serial dilutions tested by OAP-ELISA. Arrows highlight end-point titer. (C) Table summarizing the end-point titer obtained with commercial ELISA, OAPs-ELISA and CBA.</p

OAPpurification by nSEC.

<p>(A) Chromatograms obtained by S-300 (left) and S-500 (right)-based nSEC (columns 16/60). AQP4 levels in each fraction were evaluated by dot blot and data are reported on the graph displayed at the bottom of the chromatogram. Note that using S-300, AQP4 was only eluted in the first fractions (38-40ml), while using S-500, AQP4 was detected between 50 and 90ml. (B) BN-PAGE analysis of AQP4-OAP distribution in nSEC fractions. (C) SDS-PAGE analysis of the M23/M1 ratio in S-500 fractions. Note that the M23/M1 ratio reported in the histogram correlates with the OAP dimension of each fraction shown in B.</p

Analysis of OAP complexity and purification grade using 16/60 Sephacryl S-500-based nSEC.

<p>(A) Analysis of nSEC fractions obtained using 16/60 Sephacryl S-500. The E17-E30 fraction analysis was performed by SDS-PAGE followed by Coomassie blue staining and AQP4 immunoblotting. Note that 10 times more proteins were used for Coomassie blue staining than for AQP4 immunoblotting. (B) Quantification of AQP4 enrichment (ng of AQP4/ul) and purification grade (AQP4/total protein (%)) in fractions E17-E30. The red arrow indicates the fraction with the highest AQP4 purification and enrichment grade. (C) Chromatogram obtained by S-500 based nSEC and analysis. Fractions 17 and 21 were analyzed by 2DE and immunoblot (bottom). The red arrow indicates the position, in the SEC elution profile, of the OAP-containing fraction with maximal enrichment and purification grade used for 2DE.</p

OAPs-ELISA performance.

<p>(A) Scatter plot of sera tested by OAP-ELISA. Among the 67 NMO sera analyzed, 61 were found to be positive by OAP-ELISA, while in the control group 71/72 were negative by OAPs-ELISA. The cut-off lane represents the limit value for positivity/negativity. In house CBA negative were reported in red. (B) intra- and iter-assay reproducibility of OAPs-ELISA using four NMO and four control MS sera. (C) Sensitivity as a function of specificity ROC analysis curve.</p