Prior studies have reported that cystatin C concentrations are decreased in the CSF of ALS patients relative to healthy controls, but it has been unclear if these reductions in protein level result in proportional reductions in cysteine protease inhibitory activity. To address this important question, we modified a papain inhibition assay to allow for the direct measurement cystatin C functional activity in human CSF. The assay performed with low inter- and intra-assay variability, and allowed the reliable evaluation of cystatin C activity in multiple CSF samples from ALS patients and controls. Notably, this assay measures only the activity of cystatin C at its primary active site, which interacts with papain-like cysteine proteases. Therefore, the experimental findings of this study only apply to the activity of cystatin C against its primary ligands, and do not address its interactions with legumain-like cysteine proteases.
As expected, cystatin C concentration demonstrated a strong, direct relationship with the magnitude of its inhibitory activity (Figure 2). This confirms that CSF cystatin C is biologically active against papain-like cysteine proteases, and that there does not appear to be significant variability in the activity state of the protein among individual subjects.
Cystatin C concentration also demonstrated a strong, indirect relationship with the overall activity of the papain assay (Figure 2). This indicates that CSF induces an overall inhibitory effect on papain-mediated protease activity, and that this effect is highly dependent upon cystatin C concentration. Additionally, the strength of the correlations between cystatin C concentration and both its own activity (Figure 2B) and total papain assay activity (Figure 2A) were nearly identical in magnitude (r of 0.883 and -0.861, respectively). This, along with Table 2 data indicating that cystatin C antibody can completely block papain inhibition in CSF, suggests that any other endogenous papain-like cysteine proteases and alternate cysteine protease inhibitors that are present in human CSF contribute only minimally to the measured assay activity. Therefore, cystatin C is likely the dominant cysteine protease inhibitor in human CSF, and plays a critical role in the regulation of cysteine protease-mediated proteolysis in this setting. While prior studies have shown that cystatin C protein levels are enriched in the CSF versus the blood and the protein is often detected in proteomic studies of the CSF [16, 17], our data suggests it is the primary cysteine protease inhibitor in the CSF. It should be noted that if cystatin C concentrations approach saturating concentrations for their selected cysteine proteases in the CSF, then small changes in concentration between individuals or subject groups may not translate to significant physiological changes in inhibitory activity.
When the data were divided into diagnostic groups, comparable correlations were maintained between cystatin C concentration and both its own activity and total papain assay activity in each group (Table 3). Activity ratios were then calculated for each patient as the total cystatin C activity divided by the total cystatin C concentration, and the resulting mean values were nearly identical between the three experimental groups (Tables 3 and 4). However the large intragroup variations suggest that cystatin C may not be useful as an individual biomarker for ALS but could be useful in a panel of disease biomarkers.
Reduced CPI activity within the CSF of ALS or other neurologic disease patients may contribute to either toxic or protective pathways related to disease pathogenesis. Cystatin C has been reported to exhibit neurotoxic effects when injected into the rodent brain , and when directly applied to primary neuronal cultures . However, cystatin C also exhibits neuroprotective effects both in vivo and in vitro, and potential neuroprotective pathways of cystatin C have been more thoroughly characterized . The protective function that appears most relevant to ALS is the regulation of extracellular cathepsins and calpains, which are released as part of physiological processes or in response to CNS damage or stress. The levels of these proteases may be elevated in ALS and other diseases due to increased expression [23, 24], secretion by activated microglia , and release by dying neurons [24, 26–29]. Therefore, the reduced CSF cystatin C activity in ALS may be inadequate to counteract the apparent increase in protease activity, resulting in protease-mediated CNS damage.
Extracellular cystatin C also may be internalized by motor neurons and/or glial cells, and subsequently affect intracellular processes. Efficient cystatin C uptake has been demonstrated with multiple non-neuronal human cell lines . If this process also occurs in the human CNS, reduced extracellular cystatin C concentration could result in reduced uptake and abnormal deficiencies in intracellular cystatin C activity. Intracellular cathepsins and calpains both appear to be up-regulated in ALS, and can contribute to the induction of apoptosis [23, 24]. Therefore, deficiencies in intracellular cystatin C could potentially lead to apoptosis through the loss of a protective mechanism. Alternatively, reduced extracellular cystatin C levels may reflect increased cellular uptake in response to increased intracellular levels of cathepsins and calpains.