Characterizing Single-Cell Differences in Aquaporin-4 and Glutamate Transporter-1 Between Control and hSOD1G93A Astrocytes Using Integrated Epifluorescence and Structured Illumination Microscopy.

Astrocytes, the abundant glial cells of the central nervous system (CNS), maintain water and glutamate homeostasis through aquaporin-4 (AQP4) and glutamate transporter-1 (EAAT2). In the hSODG93A animal model of amyotrophic lateral sclerosis, astrocytes exhibit alterations in these homeostatic proteins. AQP4 and EAAT2 changes are observed in both hSODG93A-expressing astrocytes in the CNS and in cell cultures. Here, we provide a detailed analysis of differences between cultured control and hSOD1G93A astrocytes in AQP4 and EAAT2 fluorescence patterns, subcellular localization, and spatial overlap, using side-by-side epifluorescence and structured illumination microscopy modes of a custom imaging system [two-dimensional structured illumination microscopy (2D SIM)]. 2D SIM system characterization and fluorescence signal analysis demonstrated uniform epifluorescence illumination and high-contrast SIM patterns, enabling whole-cell imaging of AQP4 and EAAT2 and resolving their signals into distinct puncta in both control and hSOD1G93A astrocytes with SIM. Compared with control astrocytes, AQP4 expression increased and EAAT2 expression decreased in the plasma membrane and cytoplasm of hSOD1G93A astrocytes at both diffraction-limited and superresolution scales. Subdiffraction-scale analysis revealed differences in the spatial distributions of AQP4 and EAAT2, showing increased colocalization between these proteins in hSOD1G93A astrocytes. These findings demonstrate the utility of integrated imaging approaches for quantitative, single-cell-resolution analysis of disease-associated protein alterations in astrocytes.