Abnormal GLT-1 Trafficking in Alexander Disease Astrocytes, as shown by Total Internal Reflection Fluorescence Microscopy

Mutations in the gene for glial fibrillary acidic protein GFAP, which encodes the major intermediate filament protein of astrocytes, result in Alexander Disease (AxD), a fatal neurological disorder in which myelin and neurons are lost. The uptake of glutamate (Glu) via the major glutamate transporter (GLT-1) of astrocytes is important for controlling the extracellular concentrations of Glu, thus limiting Glu-mediated toxicity to other cell types. Previous work has defined a loss of GLT-1 in AxD and in mouse models of AxD. Effective. Glu uptake requires a high density of GLT-1 transporters localized to the plasma membrane, but the direct visualization of GLT-1 trafficking in astrocytes has been a major challenge. Here, we describe an optical imaging approach, based on total internal reflection fluorescence, to examine the dynamic remodeling of RFP-GLT-1 fusion protein at the cell surface of live astrocytes on a timescale of minutes. Quantification of the density, size and morphogenesis of RFPGLT-1 proteins in astrocytes expressing the R239C GFAP mutant revealed a significantly reduced number, size and motile redistribution of GLT-1 proteins compared to the wild type, particularly at transient, spine-like membrane protrusions of the R239C astrocytes. This suggests dysfunctional Glu transport may be due, in part, to a defective membrane organization of GLT-1. Our study provides the first structural insights into the intercellular mechanism underlying an astrocytic encephalopathy caused by a mutation in GFAP that compromises survival of their neighbors.