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.