Gene deletion of cystatin C aggravates brain damage following focal ischemia but mitigates the neuronal injury after global ischemia in the mouse

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Abstract—

Cystatin C is distributed in all human tissues and fluids with a particular abundance in the cerebrospinal fluid. Cystatin C is a strong endogenous inhibitor of lysosomal cysteine proteases, such as cathepsin B, L, H and S, that are involved in various biological processes such as degradation of cellular proteins and regulation of enzymes, as well as in pathological processes. Pharmacological inhibition of cathepsins has been shown to reduce neuronal damage after brain ischemia, suggesting that cystatin C is an endogenous neuroprotectant. Cystatin C has also amyloidogenic properties and is co-localized with β-amyloid in degenerated neurons in Alzheimer's disease, suggesting a role in neuronal degeneration. To test the hypothesis that endogenous cystatin C is neuroprotective during brain ischemia, global and focal brain ischemia was induced in mice with the cystatin C gene knocked out. Following focal ischemia, larger brain infarcts were found in cystatin C knockout mice, probably due to a reduced inhibition of the cathepsins during ischemia. In contrast, brain damage after global ischemia was diminished in cystatin C knockout mice, suggesting that cystatin C has an aggravating effect on selective neuronal damage after global ischemia.

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Animals

Male mice, C57BL/6J/DBA of a mixed background strain with a deficient gene for CC, described previously (Huh et al., 1999), were used. CC knockout (ko) and wild-type (wt) animals were not littermates, but generated from two separate lines bred in parallel. The gene for the agouti color is co-localized with CC ko and therefore ko animals were agouti colored and wt mice were black. Animals used for the experiments were 2–6 months old, and housed under diurnal light conditions with free access to

Absence of CC protein in CC deficient mice

CC protein in brains from CC ko and wt mice was quantified by ELISA. Brain from wt animals (n=11) contained 280±11 ng CC/mg protein, while no measurable quantity of CC protein was found in CC ko animals (n=11). Similarly, using immunohistochemistry, CC protein could be found in all cortical neurons in wt (n=5), while no immunostaining was seen in CC ko mice (n=3; Fig. 1A, B).

Focal ischemia-induced damage increases in CC ko mice

Forty minutes of MCA occlusion led to infarction in both cortex and striatum. The infarct volume was significantly larger

Discussion

The inhibition of CC gene transcription and the lack of CC protein in the CC ko mice has earlier been demonstrated (Huh et al., 1999), and we now confirm the absence of the CC protein in the brain by immunohistochemistry and ELISA. The absence of CC did not significantly affect cortical CBF, during and after ischemia.

We found that the effect of ischemia on neuronal death was opposite in the two ischemic models. Following occlusion of the MCA, infarct size increased in the CC ko mice compared

Acknowledgments

The Swedish Science Council (Project No. 08644) and the Bergendahl foundation supported this work. The authors are grateful to Johan Wasselius for help with the immunohistochemistry.

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