Fig. 9
From: Lysosomal iron modulates NMDA receptor-mediated excitation via small GTPase, Dexras1
Lysosomes are the source of iron involved with regulating NMDA-R function. a. Lysosomal subcellular fraction was isolated and immunoblotted for ACBD3 and Dexras1. Lysosomal specific marker (Rab7) was only detected in the lysosome fraction (L) but not in the cytoplasm fraction (C). b. Confocal image shows that ACBD3 is co-localized to lysosome marker LAMP-2. c. In Calcein-AM treated slices, PIH is able to de-quench Fe2+ from Calcein in CA1, and this effect is blocked by the DMT1 antagonist 10 μM ebselen and as well as 5 mM NH4Cl, which collapses the lysosome proton gradient. d. VSD imaging of CA1 shows that there is no increase in response to stimulation following PIH application when the lysosome is perturbed with NH4Cl. To the right is an averaged response of pixel intensities over time from a standardized region of CA1 in both Control (black) and PIH (red). e. There is no enhancement of the response following PIH application when slices are incubated with NH4Cl,(n = 6 slices; (p = 0.4) of with Bafilomycin, which also perturbs lysosomal release of iron (n = 9; p = 0.21). f. Western blot analysis of brain homogenates before and after PIH treatment when the brains were pre-incubated with NH4Cl. Collapsing the lysosomal proton gradient abolishes the phosphorylation of Src