| Summary: | Our previous studies suggest that a failure to degrade aggregated Aβ1-42 in late endosomes or secondary lysosomes is a mechanism that contributes to intracellular accumulation in Alzheimer's disease. In this study, we demonstrate that cultured primary neurons are able to internalize soluble Aβ1-42 from the culture medium and accumulate inside the endosomal/lysosomal system. The intracellular Aβ1-42 is resistant to protease degradation and stable for at least 48 h within the cultured neurons. Incubation of cultured neurons with a cytotoxic concentration of soluble Aβ1-42 invokes the rapid free radical generation within lysosomes and disruption of lysosomal membrane proton gradient which precedes cell death. The loss of lysosomal membrane impermeability is only specific to the Aβ1-42 isoform since incubation of cells with high concentrations of Aβ1-40 has no effect on lysosomal hydrolase release. To further support the role of lysosomal membrane damage in Aβ-mediated cell death, we demonstrate that photodisruption of acridine orange (AO)-loaded lysosomes with intense blue light induces a relatively rapid synchronous lysosomal membrane damage and neuronal death similar to that observed as a result of Aβ exposure. AO leaks quickly from late endosomes and lysosomes and partially shifts the fluorescence from an orange fluorescence to a diffuse, green cytoplasmic fluorescence. Such AO relocalization is due to an initial disruption of the lysosomal proton gradient, followed by the release of lysosomal hydrolases into the cytoplasmic compartment. Treatment of cells with either the antioxidant n-propyl gallate or lysosomotropic amine (methylamine) partially blocks the release of lysosomal contents suggesting that this AO relocalization is due to lysosomal membrane oxidation. Based on these findings, we propose that the cell death mediated by the soluble Aβ may be fundamentally different from the cell loss observed following extracellular Aβ deposition.
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