Abstract
BackgroundCystatin C is a constitutively expressed and abundant cysteine protease inhibitor within the cerebrospinal fluid (CSF). Recent studies have reported a significant reduction in cystatin C concentration in the CSF of patients with amyotrophic lateral sclerosis (ALS) and several other neurodegenerative diseases, relative to healthy controls. Cystatin C can exhibit both neuroprotective and neurotoxic properties, suggesting that altered CSF cystatin C concentrations could potentially impact the pathogenesis or progression of these disorders. However, it is unclear if alterations in cystatin C concentration result in physiologically relevant differences in its functional activity within the CSF. Measurements of the cysteine protease inhibitory activity of cystatin C within the CSF have not been reported, and the relationship between CSF cystatin C concentration and activity levels in different disease contexts has not been investigated.MethodsWe used a papain inhibition assay to evaluate the total cystatin C activity in CSF samples from 23 ALS patients, 23 healthy controls, and 23 neurological disease controls. Cystatin C concentrations in these samples were previously measured by ELISA. Correlations between cystatin C concentration and activity were assessed with nonparametric statistics. Activity ratios were compared among diagnostic groups using both one-way ANOVA and repeated measures statistics.ResultsTotal cystatin C activity was found to be directly proportional to its protein concentration in all subjects, and cystatin C activity was not altered in ALS patients. In addition, our data suggest that cystatin C is the predominant cysteine protease inhibitor in human CSF.ConclusionsOur data demonstrate the successful measurement of the functional activity of cystatin C in the CSF, and show that total cystatin C activity can be inferred from its total protein concentration. Our results also suggest that cystatin C is the major cysteine protease inhibitor in human CSF and altered CSF cystatin C concentration may play a role in the pathobiology of ALS and other neurological diseases.
Highlights
Cystatin C is a constitutively expressed and abundant cysteine protease inhibitor within the cerebrospinal fluid (CSF)
We found that the functional activity of cystatin C within the CSF is directly related to its protein concentration, and that reduced cystatin C protein levels in the CSF of amyotrophic lateral sclerosis (ALS) patients likely correspond to reduced cysteine protease inhibitory activity
There are several challenges when using this assay to measure the activity of a specific cysteine protease inhibitor (CPI) within a biofluid sample, such as CSF
Summary
Cystatin C is a constitutively expressed and abundant cysteine protease inhibitor within the cerebrospinal fluid (CSF). Cystatin C is a low molecular weight cysteine protease inhibitor that modulates protein degradation and cellmatrix interactions [1]. This protein binds with high affinity to papain, cathepsins B, H and L, calpain, and dipeptidyl peptidase, regulating both intracellular and extracellular cysteine proteases [2]. Cystatin C is 5-fold more concentrated in the CSF than in the bloodstream [3] This suggests that cystatin C may have important functions within the CSF relating to the modulation of protein degradation and, potentially, the regulation of multiple signaling pathways [4]
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