Age-related lysosomal alterations perturb intracellular cobalamin trafficking
Previous data indicates lysosomes become dysfunctional in ageing post-mitotic cells. As transit through lysosomes is essential for utilization of cobalamin (Cbl), we proposed that ageing processes (lipofuscin accumulation, altered lysosomal pH and protease activity) impair intracellular Cbl transport. Objective. To perturb lysosome function in vitro and assess the impact on intracellular [57Co]Cbl transport. Design. Human HT1080 fibroblasts and SH-SY5Y neurons were treated with either chloroquine (to increase lysosomal pH), leupeptin (to inhibit lysosomal proteases) or lipofuscin (to induce lysosomal lipofuscin loading). Cells labelled with [57Co]cyanoCbl were lysed and fractionated and [57Co] was measured in lysosomal fractions by gammacounting. Results. As a percentage of total cellular [57Co]Cbl, fibroblast lysosomal [57Co]Cbl levels increased from 6.0 + 0.1% to 23.0 + 0.8% after chloroquine treatment, and to 19.1 + 0.7% after leupeptin treatment. Lysosomal [57Co]Cbl was ~ doubled to 11.8% of total cellular [57Co]Cbl after treatment with lipofuscin, and it is noteworthy that this was under conditions where only ~10% of the cells were significantly loaded with lipofuscin as detected by flow cytometry. Similar results were obtained in experiments using SH-SY5Y neurons; e.g., lysosomal [57Co]Cbl levels were increased 12-fold with chloroquine treatment. Taken together, these data suggest that Cbl may become trapped in lysosomes under pathophysiological conditions that impair lysosomal function in ageing and neurodegenerative diseases. This is predicted to increase cellular levels of toxic metabolites homocysteine and methylmalonic acid due to diminished supply of methyl-Cbl to cytosolic methionine synthase and of 5-deoxyadenosyl-Cbl to mitochondrial methylmalonyl-coenzyme A mutase. Conclusions: These studies provide evidence that age-related lysosomal dysfunction significantly inhibits Cbl transport from lysosomes.