[9]), huntingtin (IVLD, [10]), DNA Topoisomerase I (PEDD, [11]), AP-2 alpha (DRHD, [12]), Periplakin (TVAD, [13]), FAK (VSWD, [14]) and TGEV (VVPD, [15]). Caspase-6 has garnered much attention recently since it has been shown that it is involved in the developmental pruning of axons [16,17], and it has been suggested that similar pathways might erroneously be activated in neurodegenerative disorders such as Alzheimer’s (AD) and Huntington disease (HD) [16,18]. The presence of activated caspase-6 and cleavage of caspase-6 substrates is indeed a hallmark of AD, HD and cerebral ischemia, and has been shown in a number of different animal models and patient brain tissue [18,19,20,21,22]. To assess caspase-6 activity in cell and tissue samples, peptide substrates or inhibitors need to be titrated accurately to yield meaningful results, since the peptide substrate commonly used to assess caspase-6 activity, VEID, can be cleaved by other caspases as well as the proteasome when used at too high concentrations [23,24]. In addition, even low concentrations of a VEID substrate can lead to inaccurate results if the relative amount of other proteolytic activity in the sample is significantly higher than that of caspase-6 due to small differences in kcat/Km [4]. A recently developed method using the biotinylated caspase inhibitor zVAD is significantly more specific, but to achieve this specificity, immuno- precipitation and subsequent detection of the active caspase by Western blotting is required [23]. For a more quantitative way of assessing caspase-6-specific activity, we have developed a novel method based on the cleavage of lamin A [24]. Here we show that this method is sensitive, specific and allows for the detection of caspase-6 activity in complex samples. The assay quantitatively measures caspase-6 activity and can be useful for investigating caspase-6 biology as well as the screening and intracellular efficacy assessment of caspase-6 inhibitors. Results In order to develop an activity assay that is more specific for caspase-6 than available methods relying on the cleavage of the VEID peptide, we decided to investigate the cleavage of known caspase-6 substrates. To be useful for the measurement of caspase- 6 activity, a substrate would ideally be as specific as possible. In particular it should not be cleaved by other proteases activated during apoptosis and be cut by caspase-6 in detectable quantities. The protein substrate should be easy to purify and thus available in larger amounts, and existing neo-epitope antibodies allowing for the specific detection of the cleavage fragment would be an advantage. The nuclear lamin proteins were among the first caspase-6 substrates characterized [25], and knockdown studies strongly suggest that their cleavage during apoptosis is highly dependent on the presence of caspase-6 [24]. Furthermore, purified lamin A as well as a variety of neo-epitope antibodies against the cleavage fragments are readily available, and high stability of the cleavage fragments without further degradation during the apoptotic process has been described [26] , which should facilitate their detection. The VEID peptide sequence commonly used in caspase-6 substrates and inhibitors is derived from the lamin cleavage site, but since protein context is known to change the kinetics and specificity of proteolytic events, we decided to compare the cleavage of the VEID peptide to that of the full-length lamin A protein. The lamin A protein is a more specific substrate than its VEID peptide We therefore decided to investigate whether lamin A is a specific substrate for caspase-6. In particular, we were interested in Caspase-6 Activity Assessment in Cell Culture the ability of other executioner caspases to cleave lamin A, since these are likely to show high activity in apoptotic extracts where accurate quantification of caspase-6 activity will be of interest. We first subjected the Ac-VEID-AFC substrate to digestion by caspases -3, -6 and -7, respectively. The amount of active enzyme used in each reaction was normalized to the concentration of active sites in the sample as determined by titration against the irreversible inhibitor zVAD-fmk (Fig. S1). As expected, the VEID peptide substrate, although it is best processed by caspase-6, still shows significant cleavage by caspase-3 and -7 (Fig. 1A) at high substrate concentrations (100 mM). Lower substrate concentrations can be used to achieve greater specificity, however, the difference in kcat/Km for VEID is less than 3 fold between caspase -3 and -6 [4]. This results in a small concentration window that can be exploited to achieve selectivity for caspase-6, if the same amounts of active caspase-3 and -6 are present. However, this window will be lost if the sample contains a higher concentration of active caspase-3 than caspase-6. As shown in Figure 1B, an 8 fold molar excess of caspase-3 over caspase-6 is enough to lead to a significantly higher signal from the non-specific caspase-3, making VEID-based assays problematic for the use in apoptotic samples or other cell and tissue lysates with high levels of active caspase-3. Lamin A, on the other hand, only showed proteolytic processing when incubated with recombinant active caspase-6, not with the corresponding amounts of caspases -3 and -7 at concentrations up to 300 mM (Fig. 1C). We therefore focussed on lamin A as a substrate for the development of a specific caspase-6 activity assay. To confirm the selectivity of lamin cleavage by caspase-6, we made use of mouse embryonic fibroblast (MEF) cells generated from wild-type and caspase-62/2 (C6wt and C6ko) mice [27]. These cells only differ in their expression of caspase-6 (Fig. 2A), making them an ideal system to study the specificity of caspase-6 substrates. Apoptosis was induced by the addition of 50 nM staurosporine to the culture medium, and the activation of caspase-6 was monitored over time by assessing the cleavage of endogenous lamin A via Western blotting (Fig. 2B) or with the Ac- VEID-AFC peptide substrate (Fig. 2F). The antibody used to detect full length lamin A at 70 kDa cross-reacts with the closely related lamin C protein (60 kDa, [28]), and the N-terminal fragments of both cleaved lamin A and C are detected at the same size of 28 kDa (Fig. 2B+E). Furthermore, using a cell line that does not express caspase-3 but expresses caspase-6 (Fig. 2C), we found that the lamin proteins are still processed after the induction of apoptosis with camptothecin (Fig. 2D), confirming the require- ment of caspase-6 but not caspase-3 for this process. An increase in VEID cleavage over time was observed for both C6wt and C6ko cell lines (Fig. 2F). For C6wt cells, significant increases over baseline were observed at all time points, whereas the C6ko cells show lower VEID processing initially with a non- significant increase in the first 2 h of treatment. However, at later timepoints C6ko cells exhibit similar levels of VEID cleavage as C6wt with a highly significant increase over non-treated controls (Fig. 2F), suggesting that other proteases make up for a large proportion of the VEID cleavage activity in apoptotic extracts. These results indicate that the lamin protein substrates are much more specific for cleavage by caspase-6 than the VEID peptide. Development of an electrochemiluminescence-based ELISA method for the quantitative assessment of cleaved lamin A In order to accurately quantitate the amount of cleaved lamin A generated by caspase-6, we turned to an ELISA-based assay format using the MesoscaleH platform, which allows for a fast and sensitive detection with minimal background using electrochemi- PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27680