Skip to main content
placeholder image

Age-related lysosomal alterations perturb intracellular cobalamin trafficking

Conference Paper


Abstract


  • 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.

Publication Date


  • 2013

Citation


  • Zhao, H. & Garner, B. (2013). Age-related lysosomal alterations perturb intracellular cobalamin trafficking. 33rd Meeting of the Australian Neuroscience Society: Program, Abstracts & List of Registrants (pp. 190-190). Australia: ANS.

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/912

Start Page


  • 190

End Page


  • 190

Abstract


  • 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.

Publication Date


  • 2013

Citation


  • Zhao, H. & Garner, B. (2013). Age-related lysosomal alterations perturb intracellular cobalamin trafficking. 33rd Meeting of the Australian Neuroscience Society: Program, Abstracts & List of Registrants (pp. 190-190). Australia: ANS.

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/912

Start Page


  • 190

End Page


  • 190