RESUMEN
Aquaporin-4 (AQP4) is a predominant water channel in the central nervous system. It regulates water movement in the brain and has been suggested to play critical roles in various pathological conditions. However, the molecular mechanisms underlying its regulation are not yet well understood. In this study, we biochemically characterized AQP4 in the brain using acute cortical brain slices prepared from mice. Using biochemical fractionation, we found that AQP4 is enriched in the detergent-resistant membrane (DRM) fraction that is not soluble in 1% Triton X-100. In contrast, ß-dystroglycan and syntrophin, which are part of the dystrophin complex in the brain, primarily reside in the non-DRM fraction. DRM enrichment of AQP4 is insensitive to cholesterol depletion, suggesting that it is not tightly associated with lipid rafts. Furthermore, AQP4 in the DRM fraction is more enriched in the M23 isoform than in the non-DRM fraction. Finally, by employing oxygen-glucose deprivation (OGD), an in vitro model of ischemia, we examined the molecular changes of AQP4. Under OGD conditions, a reduction in AQP4 in the DRM fraction was observed before the total AQP4 protein level dropped. Our data therefore highlight the characteristics of two pools of AQP4 that are distinctly regulated under ischemic conditions.
Asunto(s)
Acuaporina 4/metabolismo , Bioquímica/métodos , Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Fraccionamiento Químico/métodos , Animales , Células CHO , Cricetulus , Glucosa , Técnicas In Vitro , Ratones , OxígenoRESUMEN
Purpose: To observe the effect of soya yoghurt (Soyghurt), which is high in flavonoid substance, on the expression of preeclampsia biomarkers (sFLT-1 and PLGF) on preeclampsia serum-induced trophoblast primary cell culture isolated from placental tissue. Methods: The trophoblast primary culture was induced by preeclampsia serum (10%). The Soyghurt treatment was performed with 2.5%, 5%, and 7.5% Soyghurt supernatant concentrations in culture media. The expression of preeclampsia markers, sFLT-1 and PLGF, were evaluated using ELISA. Results: Expression of sFLT-1 on preeclampsia-induced cell culture treated with Soyghurt was significantly lowered compared to the untreated group (p<0.01). However, no significant difference was observed in the PLGF levels of all groups induced by preeclampsia serum (p>0.05). Conclusion: This study demonstrates the potential effect of Soyghurt's in balancing preeclampsia marker expression by inhibiting the expression of sFLT-1 in preeclampsia serum -induced trophoblast cells.
RESUMEN
Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.
RESUMEN
Aquaporin-4 (AQP4), the most abundant water channel in the brain, plays a central role in water homeostasis, neuronal activity, and migration of astrocytes in the central nervous system. Recent studies have demonstrated that AQP4 is a target of an autoantibody specifically detected in an autoimmune neurologic disease called neuromyelitis optica. Here we have generated a monoclonal antibody (MAb) against the C-terminal region of AQP4 using a baculovirus expressing mouse AQP4 as an immunogen. This antibody (clone E5206) recognized both human and mouse AQP4s in a denaturing condition and was able to precipitate AQP4 from cell lysates of CHO cells stably expressing AQP4. Western blot analysis using deletion mutants revealed that the epitope was located within a region between Asp(303) and Leu(320) in the C-terminal tail of AQP4. Although clone E5206 could not be used for immunostaining when cells or tissues were fixed with 4% paraformaldehyde or 10% formalin, it could be used when cells were fixed with 10% trichloroacetic acid and when a formalin-fixed tissue section was pretreated with antigen-retrieval reagents. This MAb can be a valuable tool for analysis of AQP4 in a variety of physiological and pathophysiological contexts, in human tissues and organs as well as in rodent models, both in vitro and in vivo.