Measurement of cystatin C functional activity in the cerebrospinal fluid of amyotrophic lateral sclerosis and control subjects

Abstract

Background: Cystatin 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.

Methods: We 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.

Results: Total 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.

Conclusions: Our 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.

Figure 1

Depletion of cystatin C from CSF by immunoprecipitation. Lanes 1–4: Increasing amounts of anti-cystatin C polyclonal antibody (0–1.6 μg antibody) was added to CSF for immunoprecipitation as described in Methods. The resulting immunoprecipitate was analysed by western blot for cystatin C. Lane 1: 1.6 μg antibody (IP1); Lane 2: 0.8 μg antibody (IP2); Lane 3: 0.4 μg antibody; (IP3) Lane 4: 0 μg antibody (IP4). The remaining column flow-through from each sample was then used to repeat the cystatin C immunoprecipitation using 0.8 μg antibody to examine efficiency of cystatin C removal during the initial immunoprecipitation. Lane 5: Flow-through from IP1; Lane 6: Flow-through from IP2; Lane 7: Flow-through from IP3; Lane 8: Flow-through from IP4; Lane 9: Human cystatin C purified protein as a positive control from a separate gel.

Figure 2

Correlation of cystatin C concentration with papain inhibition assay activity and cystatin C activity. (A) Total assay activity was indirectly related to cystatin C concentration, and the relationship was statistically significant (p < 0.001, Spearman r = -0.861). (B) Total cystatin C activity was directly related to its own concentration, and the correlation was strong and significant (p < 0.001, Spearman r = 0.883).