Abstract
Neuronal cell culture models have been used to demonstrate the protective effects of cystatin C against a variety of insults, including the toxicity induced by oligomeric and fibrillar amyloid β (Aβ). Here, we describe assays quantifying cystatin C protective effects against cytotoxicity induced by nutrient deprivation, oxidative stress, or cytotoxic forms of Aβ. Three methods for the evaluation of either cell death or cell survival are described: measurement of metabolic activity, cell death, and cell division. The cell culture models used are murine primary cortical neurons and murine primary cerebral smooth muscle cells. The effects of exogenously applied cystatin C are studied by comparing the viability of nonstressed control, stressed control, and cystatin C-treated stressed cells. The effect of endogenous level of cystatin C expression is studied by comparing stressed primary cells isolated from brains of cystatin C transgenic, cystatin C knockout, and wild-type mice.
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References
Sastre, M., Calero, M., Pawlik, M., Mathews, P. M., Kumar, A., Danilov, V., Schmidt, S. D., Nixon, R. A., Frangione, B., and Levy, E. (2004) Binding of cystatin C to Alzheimer’s amyloid β inhibits amyloid fibril formation. Neurobiol Aging 25, 1033–1043.
Kaeser, S. A., Herzig, M. C., Coomaraswamy, J., Kilger, E., Selenica, M. L., Winkler, D. T., Staufenbiel, M., Levy, E., Grubb, A., and Jucker, M. (2007) Cystatin C modulates cerebral β-amyloidosis. Nat Genet 39, 1437–1439.
Mi, W., Pawlik, M., Sastre, M., Jung, S. S., Radvinsky, D. S., Klein, A. M., Sommer, J., Schmidt, S. D., Nixon, R. A., Mathews, P. M., and Levy, E. (2007) Cystatin C inhibits amyloid-β deposition in Alzheimer’s disease mouse models. Nat Genet 39, 1440–1442.
Selenica, M. L., Wang, X., Ostergaard-Pedersen, L., Westlind-Danielsson, A., and Grubb, A. (2007) Cystatin C reduces the in vitro formation of soluble Aβ1-42 oligomers and protofibrils. Scand J Clin Lab Invest 67, 179–190.
Tizon, B., Ribe, E. M., Mi, W., Troy, C. M., and Levy, E. (2010) Cystatin C protects neuronal cells from amyloid β-induced toxicity. J Alzheimers Dis 19, 665–894.
Tizon, B., Sahoo, S., Yu, H., Gauthier, S., Kumar, A. R., Mohan, P., Figliola, M., Pawlik, M., Grubb, A., Uchiyama, Y., Bandyopadhyay, S., Cuervo, A. M., Nixon, R. A., and Levy, E. (2010) Induction of autophagy by cystatin C: a mechanism that protects murine primary cortical neurons and neuronal cell lines. PLoS One 5, e9819.
Deng, A., Irizarry, M. C., Nitsch, R. M., Growdon, J. H., and Rebeck, G. W. (2001) Elevation of cystatin C in susceptible neurons in alzheimer’s disease. Am J Pathol 159, 1061–1068.
Palm, D. E., Knuckey, N. W., Primiano, M. J., Spangenberger, A. G., and Johanson, C. E. (1995) Cystatin C, a protease inhibitor, in degenerating rat hippocampal neurons following transient forebrain ischemia. Brain Res 691, 1–8.
Pirttila, T. J., and Pitkanen, A. (2006) Cystatin C expression is increased in the hippocampus following photothrombotic stroke in rat. Neurosci Lett 395, 108–113.
Huh, C. G., Hakansson, K., Nathanson, C. M., Thorgeirsson, U. P., Jonsson, N., Grubb, A., Abrahamson, M., and Karlsson, S. (1999) Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene. Mol Pathol 52, 332–340.
Pawlik, M., Sastre, M., Calero, M., Mathews, P. M., Schmidt, S. D., Nixon, R. A., and Levy, E. (2004) Overexpression of human cystatin C in transgenic mice does not affect levels of endogenous brain amyloid β peptide. J Mol Neurosci 22, 13–18.
Abrahamson, M., Barrett, A. J., Salvesen, G., and Grubb, A. O. (1986) Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem 261, 11282–11289.
Yamada, T., Mukaiyama, I., Miyake, N., and Igari, J. (2002) Measurement of cystatin C in cerebrospinal fluid. Rinsho Byori 50, 613–617.
Jones, K. H., and Senft, J. A. (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem 33, 77–79.
Soames, A. R., Lavender, D., Foster, J. R., Williams, S. M., and Wheeldon, E. B. (1994) Image analysis of bromodeoxyuridine (BrdU) staining for measurement of S-phase in rat and mouse liver. J Histochem Cytochem 42, 939–944.
Perry, S., Norman, J., Litzburg, A., and Gelbard, H. (2004) Antioxidants are required during the early critical period, but not later, for neuronal survival. J Neurosci Res 78, 485–492.
Acknowledgments
This work was supported by grants from the NIA (AG017617) and the Alzheimer’s Association (IIRG-07-59699).
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Gauthier, S.A., Tizon, B., Sahoo, S., Levy, E. (2012). In Vitro Assays Measuring Protection by Proteins such as Cystatin C of Primary Cortical Neuronal and Smooth Muscle Cells. In: Sigurdsson, E., Calero, M., Gasset, M. (eds) Amyloid Proteins. Methods in Molecular Biology, vol 849. Humana Press. https://doi.org/10.1007/978-1-61779-551-0_19
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DOI: https://doi.org/10.1007/978-1-61779-551-0_19
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