P2R Inhibitors Prevent Antibody-Mediated Complement Activation in an Animal Model of Neuromyelitis Optica : P2R Inhibitors Prevent Autoantibody Injury.

Purinergic 2 receptors (P2Rs) contribute to disease-related immune cell signaling and are upregulated in various pathological settings, including neuroinflammation. P2R inhibitors have been used to treat inflammatory diseases and can protect against complement-mediated cell injury. However, the mechanisms behind these anti-inflammatory properties of P2R inhibitors are not well understood, and their potential in CNS autoimmunity is underexplored. Here, we tested the effects of P2R inhibitors on glial toxicity in a mouse model of neuromyelitis optica spectrum disorder (NMOSD). NMOSD is a destructive CNS autoimmune disorder, in which autoantibodies against astrocytic surface antigen Aquaporin 4 (AQP4) mediate complement-dependent loss of astrocytes. Using two-photon microscopy in vivo, we found that various classes of P2R inhibitors prevented AQP4-IgG/complement-dependent astrocyte death. In vitro, these drugs inhibited the binding of AQP4-IgG or MOG-IgG to their antigen in a dose-dependent manner. Size-exclusion chromatography and circular dichroism spectroscopy revealed a partial unfolding of antibodies in the presence of various P2R inhibitors, suggesting a shared interference with IgG antibodies leading to their conformational change. Our study demonstrates that P2R inhibitors can disrupt complement activation by direct interaction with IgG. This mechanism is likely to influence the role of P2R inhibitors in autoimmune disease models and their therapeutic impact in human disease.

Author Correction: GSK3ß and ERK regulate the expression of 78 kDa SG2NA and ectopic modulation of its level affects phases of cell cycle.

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GSK3ß and ERK regulate the expression of 78 kDa SG2NA and ectopic modulation of its level affects phases of cell cycle.

Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3ß by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3ß. In cells depleted from SG2NA by shRNA, the levels of pGSK3ß and pERK are reduced, suggesting that these kinases and SG2NA regulate each other's expression.

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress.

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.

Tissue specific expression of SG2NA is regulated by differential splicing, RNA editing and differential polyadenylation.

SG2NA belongs to a three member Striatin subfamily of WD-40 repeat superfamily. It has multiple protein-protein interaction domains that are involved in the assembly of supra-molecular signaling complexes. Earlier we had demonstrated that there are at least five variants of SG2NA, generated by alternative splicing. We now demonstrate that a 52kDa novel variant is generated by the editing of the transcript for the 82kDa isoform. The 52kDa protein is abundant in mouse tissues but it is barely present in immortalized cells, suggesting its role in cell differentiation. Besides splicing and editing, expression of SG2NAs in tissues is also regulated by differential polyadenylation and mRNA/protein stability. Further, the longer UTR is seen only in the brain mRNA from 1month old mouse and 8-10day old chick embryo. Like alternative splicing, differential polyadenylation of Sg2na transcripts is also conserved in evolution. Taken together, these results suggest a highly versatile and dynamic mode of regulation of SG2NA with potential implications in tissue development.