Year

2014

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences

Abstract

Vitamin B12, known as cobalamin (Cbl), is required for erythrocyte formation and DNA synthesis. It plays a crucial role in maintaining neurological function. As a coenzyme for methionine synthase and methylmalonyl-CoA mutase, Cbl utilisation depends on its efficient transit through the intracellular lysosomal compartment and subsequent delivery to the cytosol and mitochondria. Lysosomal function deteriorates in ageing and Alzheimer’s disease (AD). Although rodent studies indicate that Cbl supplementation significantly improves cognitive performance, human trials have failed to provide a consistent beneficial effect on cognitive performance with either oral or parenteral Cbl. This thesis proposes a novel hypothesis that neuropathological conditions that impair lysosomal function, such as age-related lipofuscinosis, lysosomal storage diseases, and AD, may interrupt lysosomal Cbl transport and thereby impede Cbl utilisation. The experiments will apply, for the first time, in vitro and in vivo models of ageing and AD to define how lysosomal perturbations directly affect Cbl utilisation.

To address this question, a subcellular fractionation method was developed and western blot and gamma counting techniques were used to measure organelle marker proteins and [57Co] Cbl radioactivity levels in isolated purified lysosomes, mitochondria, and cytosol that were derived from neuronal cells and mouse brain tissue. The results from cultured cells, treated with compounds to impair lysosomal protease function, revealed a ten-fold increase of [57Co] Cbl in lysosomes concomitant with reduced [57Co] Cbl levels in mitochondria and cytosol. Artificial lipofuscin was synthesized and fed into cells, which also resulted in an accumulation in lysosomal [57Co] Cbl levels. In addition, lysosomal [57Co] Cbl transport was interrupted in lysosomal glycosphingolipid storage disease cells derived from a patient with Gaucher’s disease, where lysosomal glucosylceramides had accumulated and lysosomal [57Co] Cbl levels were doubled. Furthermore, C57BL/6J wild type mice and APPxPS1 AD mice were intraperitoneally injected with [57Co] Cbl and the amount of [57Co] Cbl radioactivity in the major organs was measured. The [57Co] Cbl level in the APPxPS1 AD mouse brains demonstrated a significant increase in lysosomes and a decrease in cytosol compared to the wild type mice. These in vivo experiments were replicated using APP mutant cells treated with a proteasome inhibitor to induce lysosomal amyloid-beta accumulation. This similarly increased lysosomal [57Co] Cbl levels.

In summary, the results from the in vitro and in vivo experiments provide a detailed understanding of the impact of lysosomal dysfunction related to brain ageing and AD on lysosomal Cbl transport at the subcellular level. These results may also explain why Cbl administration has not yielded a consistent therapeutic benefit in the ageing and AD contexts. More importantly, this thesis sheds light on this crucial issue and is a step towards identifying a clinical therapeutic target to improve neuronal Cbl utilisation and thus reduce the production of neurotoxic metabolites that accumulate when the coenzyme forms of Cbl do not reach their intracellular targets.

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